Monoclonal antibodies against claudin-18 for treatment of cancer

ABSTRACT

The present invention provides antibodies useful as therapeutics for treating and/or preventing diseases associated with cells expressing CLD1 8, including tumor-related diseases such as gastric cancer, esophageal cancer, pancreatic cancer, lung cancer, ovarian cancer, colon cancer, hepatic cancer, head-neck cancer, and cancer of the gallbladder.

RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 12/096,216, now U.S. Pat. No. 8,088,913, which was filed on Oct. 14,2008 as a National Stage Entry of PCT/EP2006/011785, which was filed onDec. 7, 2006 and claimed priority benefit of European Patent ApplicationNumber 05 026 874.7, which was filed on Dec. 8, 2005. The contents ofthe above-identified applications are incorporated by reference in theirentireties.

Antibody-based therapies for cancer have the potential of higherspecificity and lower side effect profile as compared to conventionaldrugs. The reason is a precise distinction between normal and neoplasticcells by antibodies and the fact, that their mode of action relies onless toxic immunological anti-tumor mechanisms, such as complementactivation and recruitment of cytotoxic immune cells.

Targets for antibody-based therapies need to have particular qualities,which form the basis for proper discrimination between normal andneoplastic cells. Obviously, a target with either exclusive restrictionto tumor cells and entirely undetectable on normal tissues is ideal forthe development of efficient and safe antibody therapeutics. In anotheraspect, a high-level overexpression may be the basis for the therapeuticwindow and low side effects exemplified by the human epidermal growthfactor receptor type 2 (HER-2), which as a result of gene amplificationis a good target for the antibody trastuzumab (Herceptin).

Other targets for antibodies which are either already approved or inclinical development for tumor therapy have distinct qualities, whichare not based on a numeric overexpression of target molecules on tumorcells. In the case of antibodies to the proteoglycan MUC-1, a peptiderepeat epitope in the backbone of the target is underglycosylated intumor cells and thus altered to its normal counterpart. In the case ofantibodies to CD20 (rituximab), CD52 (Campath-1H) and CD22(epratuzumab), antibody targets have comparable expression levels ontumor cells and normal lymphocytes. Here, the ablation of normal cellsby the antibody is tolerable since target-negative stem cells restorethe normal lymphocyte repertoire. Other examples of differentialaccessibility of antibody targets are carcinoembryonal antigen (CEA) andcarboanhydrase IX (CA9). Both antigens are expressed on normal epitheliaof colon and kidney, respectively. However, radioactively labeledimaging antibodies do distinguish well between tumor and normal tissue,and cytotoxic antibodies are well tolerated. This is most likely due toa restricted expression of CA9 and CEA on the luminal side of normalepithelial tissue where IgG antibodies do not have access. Also antigenepithelial cell adhesion molecule (Ep-CAM) belongs to this category. Asa homotypic cell adhesion molecule for epithelial cells it is localizedin the intercellular space. Intriguingly, whereas high-affinityanti-Ep-CAM antibodies are very toxic, intermediate-affinity antibodiesare well tolerated. This suggests accessibility of the Ep-CAM target onnormal cells but also indicates that kinetics of antibody binding mayopen a therapeutic window.

One possibility is that other epithelial cell-specific proteins involvedin cell/cell adhesion may be also attractive for antibody approaches,since they may be barely accessible in well-structured epithelia toantibodies but become exposed on tumor cells. We therefore analyzedproteins involved in organizing epithelial tissue architecture for theirsuitability as targets for therapeutic antibodies. A protein, whichparticularly attracted our attention is claudin 18.

The claudin 18 (CLD18) molecule (Genbank accession number: splicevariant 1 (CLD18A1): NP_057453, NM_016369, and splice variant 2(CLD18A2): NM_001002026, NP_001002026) is an integral transmembraneprotein with a molecular weight of approximately 27.9/27.72 kD. Claudinsare integral membrane proteins located within the tight junctions ofepithelia and endothelia. Tight junctions organize a network ofinterconnected strands of intramembranous particles between adjacentcells. In tight junctions, occludin and claudins are the most prominenttransmembrane protein components. Due to their strong intercellularadhesion properties they create a primary bather to prevent and controlthe paracellular transport of solutes and restrict the lateral diffusionof membrane lipids and proteins to maintain cellular polarity. Tightjunction forming proteins are critically involved in organizingepithelial tissue architecture. We assumed that such proteins may bebarely accessible to antibodies in well-structured epithelia but becomeexposed on tumor cells.

CLD18 is a tetraspanin and has as such 4 hydrophobic regions. We havegenerated data indicating that CLD18 displays several differentconformations, which may be selectively addressed by antibodies. Oneconformation (CLD18-Conformation-1) implies, that all four hydrophobicregions serve as regular transmembrane domains (TM) and twoextracellular loops (loop1 embraced by hydrophobic region 1 andhydrophobic region 2; loop2 embraced by hydrophobic regions 3 and 4) areformed, as described for the vast majority of claudin family members. Asecond conformation (CLD18-Conformation-2) implies that, as describedfor PMP22, another member of the tetraspanin family (Taylor et al., J.Neurosc. Res. 62:15-27, 2000), that the second and third hydrophobicdomains do not fully cross the plasma membrane so that portion (loopD3)in between the first and fourth transmembrane domain is extracellular. Athird conformation (CLD18-Conformation-3) implies, a large extracellulardomain with two internal hydrophobic regions embraced by the first andfourth hydrophobic region, which serve as regular transmembrane domains.Due to the presence of classical N-glycosylation site in loopD3 theClaudin-18 topology variants CLD18 topology-2- and CLD18 topology-3harbour an additional extracellular N-glycosylation site.

Another level of complexity is added to CLD18 molecule by the presenceof two different splice variants, which are described in mouse and inhuman (Niimi, Mol. Cell. Biol. 21:7380-90, 2001). The splice variantsCLD18A1 and CLD18A2 differ in the first 21 N-terminal amino acids, whichcomprise the first TM and loop1, whereas the primary protein sequence ofthe C-terminus is identical.

CLD18A1 is selectively expressed on normal lung and stomach epithelia,whereas CLD18A2 is expressed only on gastric cells (Niimi, Mol. Cell.Biol. 21:7380-90, 2001). Most importantly, CLD18A2 is restricted to thedifferentiated short-lived cells of stomach epithelium but is devoidfrom the gastric stem cell region. Using sensitive RT-PCR, we have shownthat both variants are not detectable at all in any other normal humanorgan, but are robustly expressed in several cancer types includingstomach, esophageal, pancreatic and lung tumors as well as human cancercell lines. Expression is most prominent in the adenocarcinoma subtypesof these indications.

The molecular weight of the protein differs in some cancers and adjacentnormal tissue. The higher molecular weight protein observed in healthytissue can be transferred into the same molecular weight as observed incancer by treating tissue lysates with the deglycosylating compoundPNGase F. This suggests, that CLD18 is less N-glycosylated in cancer ascompared to its normal tissue counterpart. This structural difference islikely to give rise to an altered epitope. A classical N-glycosylationmotif is in position aa 116 within the loopD3 domain of the molecule.

The terms “CLD18” and “CLD18-variant” according to the invention shallencompass (i) CLD18-splice variants, (ii) CLD18-N-glycosylationvariants, (iii) CLD18-conformation variants, (iv) CLD18-free andhomotypically/heterotypically associated variants localized atintercellular tight junctions and (v) CLD18-cancer related andCLD18-non-cancer cell related variants.

The molecular and functional characteristics of CLD18 make this moleculea highly interesting target for antibody based cancer therapy. These arein particular (i) the absence of CLD18 from the vast majority oftoxicity relevant normal tissues, (ii) the restriction of CLD18A2variant expression to a dispersible cell population as differentiatedgastric cells, which can be replenished by target-negative stem cells ofthe stomach, (iii) hints to potential differential glycosylation betweennormal and neoplastic cells, and (iv) the presence of differentconformational topologies. Moreover, the role of CLD18 as tight junctionprotein may further contribute to a good therapeutic window. Becausetumor cells express claudins but often do not form classical tightjunctions by homotypic and heterotypic association of claudins as foundin normal epithelial tissue, tumor cells may have a considerable pool offree claudin that is amenable to extracellular antibody binding andimmunotherapy. It is possible that binding epitopes of claudins inhealthy epithelium are shielded within tight junctions from the accessby such antibodies.

The object of the invention is to provide antibodies useful for therapyof diseases wherein CLD18 is expressed, such as tumor diseases. Theantibodies described herein have also utility in diagnosing suchdiseases.

SUMMARY OF THE INVENTION

The present invention generally provides antibodies useful astherapeutics for treating and/or preventing diseases associated withcells expressing CLD18, including tumor-related diseases such as gastriccancer, esophageal cancer, pancreatic cancer, lung cancer, ovariancancer, colon cancer, hepatic cancer, head-neck cancer, and cancer ofthe gallbladder.

In one aspect the invention relates to an antibody having the ability ofbinding to CLD18 and mediating killing of cells expressing CLD18.Preferably, the antibody binds to CLD18A1 and CLD18A2 and morepreferably binds to CLD18A2 but not to CLD18A1. Preferably, antibodiesof the invention bind to and are specific for loop1 or loop2 ofCLD-conformation-1. In further preferred embodiments, the antibody ofthe invention binds to and is specific for loopD3 of CLD-conformation-2and, in particular, binds at or around a potential N-glycosylation siteat position 116 within loopD3. In further embodiments, the antibody ofthe invention is specific for the unglycosylated form of the potentialN-glycosylation site at position 116 within loopD3.

Killing of cells by the antibody of the invention is preferably inducedby binding of the antibody to CLD18 expressed by said cells, morepreferably by binding of the antibody to CLD18A2 expressed by saidcells. In one embodiment, binding of the antibody of the invention toCLD18A1 expressed by said cells does not induce killing of said cells.

The cells expressing CLD18 are preferably cancer cells and are, inparticular, selected from the group consisting of tumorigenic gastric,esophageal, pancreatic, lung, ovarian, colon, hepatic, head-neck, andgallbladder cancer cells.

Preferably the antibody of the invention mediates killing of cells byinducing complement dependent cytotoxicity (CDC) mediated lysis,antibody dependent cellular cytotoxicity (ADCC) mediated lysis,apoptosis, homotypic adhesion, and/or phagocytosis, preferably byinducing CDC mediated lysis and/or ADCC mediated lysis.

In one embodiment the antibody of the invention does not induce CDCmediated lysis of cells.

Preferably, ADCC mediated lysis of cells takes place in the presence ofeffector cells, which in particular embodiments are selected from thegroup consisting of monocytes, mononuclear cells, NK cells and PMNs, andphagocytosis is by macrophages.

The antibody of the invention may be a monoclonal, chimeric, human, orhumanized antibody, or a fragment of an antibody and may be selectedfrom the group consisting of an IgG1, an IgG2, preferably IgG2a andIgG2b, an IgG3, an IgG4, an IgM, an IgA1, an IgA2, a secretory IgA, anIgD, and an IgE antibody.

According to all aspects of the invention, CLD18 is preferably humanCLD18, preferably human CLD18A2, and CLD18A2 preferably has the aminoacid sequence according to SEQ ID NO:2 and CLD18A1 preferably has theamino acid sequence according to SEQ ID NO:8.

In particular preferred embodiments, the antibody of the invention bindsto native epitopes of CLD18 present on the surface of living cells. Infurther preferred embodiments, the antibody of the invention is specificfor cancer cells, preferably stomach cancer cells.

In certain embodiments of the invention CLD18 is expressed on thesurface of cells.

Antibodies of the invention may be obtained by a method comprising thestep of immunizing an animal with a protein or peptide having an aminoacid sequence selected from the group consisting of SEQ ID NO:2, 4, 6,16, 18, 20, 21-23, and 26-31, or an immunogenic fragment thereof, or anucleic acid or host cell expressing said protein or peptide, orimmunogenic fragment thereof. Preferably, an antibody of the inventionis specific for the aforementioned proteins, peptides or immunogenicfragments thereof.

In a particularly preferred embodiment, the antibody of the invention isproduced by a clone having the accession no. DSM ACC2737(182-D1106-055), DSM ACC2738 (182-D1106-056), DSM ACC2739(182-D1106-057), DSM ACC2740 (182-D1106-058), DSM ACC2741(182-D1106-059), DSM ACC2742 (182-D1106-062), DSM ACC2743(182-D1106-067), DSM ACC2745 (182-D758-035), DSM ACC2746 (182-D758-036),DSM ACC2747 (182-D758-040), DSM ACC2748 (182-D1106-061), DSM ACC2808(182-D1106-279), DSM ACC2809 (182-D1106-294), or DSM ACC2810(182-D1106-362).

In one embodiment the antibody of the invention is coupled to atherapeutic agent such as a toxin, a radioisotope, a drug or a cytotoxicagent.

In a further aspect the invention relates to a hybridoma capable ofproducing the antibody of the invention. Preferred hybridomas are thosehaving the accession no. DSM ACC2737 (182-D1106-055), DSM ACC2738(182-D1106-056), DSM ACC2739 (182-D1106-057), DSM ACC2740(182-D1106-058), DSM ACC2741 (182-D1106-059), DSM ACC2742(182-D1106-062), DSM ACC2743 (182-D1106-067), DSM ACC2745(182-D758-035), DSM ACC2746 (182-D758-036), DSM ACC2747 (182-D758-040),DSM ACC2748 (182-D1106-061), DSM ACC2808 (182-D1106-279), DSM ACC2809(182-D1106-294), or DSM ACC2810 (182-D1106-362).

Antibodies of the invention are designated herein by referring to thedesignation of the antibody, e.g. 182-D758-035, and/or by referring tothe clone producing the antibody, e.g. 26D12.

The invention also relates to a pharmaceutical composition comprising anantibody of the invention and/or a conjugate thereof with a therapeuticagent, and a pharmaceutically acceptable carrier.

In a further aspect the invention relates to a method of inhibitinggrowth and/or killing of a cell expressing CLD18, preferably CLD18A2,comprising contacting the cell with an effective amount of an antibodyof the invention and/or a conjugate thereof with a therapeutic agent.CLD18 is preferably expressed on the surface of said cell.

In a further aspect the invention relates to a method of treating orpreventing a disease or disorder involving cells expressing CLD18,preferably CLD18A2, comprising administering to a subject an antibody ofthe invention, a conjugate thereof with a therapeutic agent, or apharmaceutical composition comprising the antibody of the invention orthe conjugate thereof with a therapeutic agent. Preferably the diseaseor disorder is a tumor-related disease and in particular embodiments isselected from the group consisting of gastric cancer, esophageal cancer,pancreatic cancer, lung cancer, ovarian cancer, colon cancer, hepaticcancer, head-neck cancer, and cancer of the gallbladder. CLD18 ispreferably expressed on the surface of said cells.

Preferably, the antibodies of the invention have the ability todiscriminate CLD18-variants expressed by different cell types includingcancer cells and non-malignant cells. In a particularly preferredembodiment, the antibodies of the invention have the ability to bind toCLD18A2 while they do not bind to CLD18A1, or bind to CLD18A1 with alower specificity compared to the binding specificity to CLD18A2.

The term “binding” according to the invention preferably relates to aspecific binding. “Specific binding” means that an agent such as anantibody binds stronger to a target such as an epitope for which it isspecific compared to the binding to another target. An agent bindsstronger to a first target compared to a second target if it binds tothe first target with a dissociation constant (K_(D)) which is lowerthan the dissociation constant for the second target. Preferably thedissociation constant (K_(D)) for the target to which the agent bindsspecifically is more than 10-fold, preferably more than 20-fold, morepreferably more than 50-fold, even more preferably more than 100-fold,200-fold, 500-fold or 1000-fold lower than the dissociation constant(K_(D)) for the target to which the agent does not bind specifically.

The antibodies of the invention mediate killing of cells expressingCLD18, preferably CLD18A2, by binding to CLD18, preferably expressed onthe surface of said cells. In one embodiment, antibodies of theinvention induce complement dependent cytotoxicity (CDC), e.g. at leastabout 20-40% CDC mediated lysis, preferably about 40-50% CDC mediatedlysis, and more preferably more than 50% CDC mediated lysis of cellsexpressing CLD18. Such antibodies are exemplified herein by thefollowing antibodies: 37H8, 38G5, 38H3, 39F11, 61C2, 26B5, 26D12, 28D10,163E12, 175D10, 45C1, 125E1, ch-163E12, and ch-175D10. Alternatively orin addition to inducing CDC, antibodies of the invention may induceantibody dependent cellular cytotoxicity (ADCC) of cells expressingCLD18 in the presence of effector cells (e.g., monocytes, mononuclearcells, NK cells and PMNs). Such antibodies are exemplified herein by thefollowing antibodies: 37G11, 37H8, 38G5, 38H3, 39F11, 43A11, 61C2, 2685,26D12, 28D10, 42E12, 163E12, 175D10, 45C1, and 125E1. Antibodies of theinvention may have the ability to induce apoptosis of cells expressingCLD18, induce homotypic adhesion of cells expressing CLD18 and/or inducephagocytosis of cells expressing CLD18 in the presence of macrophages.The antibodies of the invention may have one or more of the abovedescribed functional properties. Preferably, antibodies of the inventioninduce CDC mediated lysis and ADCC mediated lysis of cells expressingCLD18 and more preferably induce ADCC mediated lysis of cells expressingCLD18 while they do not induce CDC mediated lysis of said cells.Exemplary target cells for antibodies of the present invention include,but are not limited to, cancer cells expressing CLD18, preferablyCLD18A2, such as tumorigenic gastric, pancreatic, esophageal and lungcancer cells. In a particular preferred embodiment, killing of cellsmediated by antibodies of the invention is CLD18A2 specific, i.e.antibodies of the invention mediate killing of cells, preferably CDCand/or ADCC mediated lysis of cells, expressing CLD18A2 but do notmediate killing of cells expressing CLD18A1 but not expressing CLD18A2.The antibodies described above may be used to mediate killing of tumorcells in the treatment or prevention of cancer such as gastric cancer,esophageal cancer, pancreatic cancer, lung cancer, ovarian cancer, coloncancer, hepatic cancer, head-neck cancer, and cancer of the gallbladder.

Antibodies of the invention may be categorized into distinct classesaccording to their binding properties and their ability to mediateeffector function on cells expressing CLD18. The antibodies of theinvention may be categorized according to their

-   -   binding properties to and/or effector functions mediated on        cells expressing either CLD18A1 or CLD18A2 (discrimination of        CLD18 splice variants),    -   binding properties to and/or effector functions mediated on        cells expressing either glycosylated or non-glycosylated CLD18        variants (discrimination between CLD18-variants with and without        N-glycosylation),    -   binding properties to and/or effector functions mediated on        either cancer cells or normal cell types (discrimination between        CLD18-variants expressed by tumor cells or normal nonmalignant        cells),    -   binding properties to CLD18-epitopes masked by the formation of        tight junctions,    -   abilities to induce aggregate formation of CLD18 on living        cells, and    -   abilities to bind a non-human CLD18 variant, particularly CLD18        variants from mice, rats, rabbits and primates.

Antibodies of the invention may have one or more of the followingproperties whereby reference is given to specific examples of antibodiesof the invention described herein (24H5, 26B5, 26D12, 28D10, 37G11,37H8, 38G5, 38H3, 39F11, 4106, 42E12, 43A11, 44E10, 47D12, 61C2, 75B8,85A3, 9E8, 19B9, 45C1, 125E1, 163E12, 166E2, 175D10, ch-43A11, ch-45C1,ch-125E1, ch-163E12, ch-166E2, ch-175D10):

-   a) binding to CLD18A2 as well as to CLD18A1 (e.g. 26D12, 28D10,    37H8, 38H3, 39F11, 61C2, and 4106)-   b) binding to CLD18A2 but not to CLD18A1 (e.g. 26B5, 37G11, 38G5,    42E12, and 43A11, 45C1, 125E1, 163E12, 166E2, 175D10, ch-43A11,    ch-45C1, ch-125E1, ch-163E12, ch-166E2, ch-175D10)-   c) binding to CLD18 naturally expressed by tumor cells but not to    CLD18 naturally expressed by non-cancer cells or tissues such as    cells of stomach and lung (e.g 26B5, 75B8, 24H5, 39F11, 45C1, 125E1,    163E12, 166E2, 175D10).-   d) mediating CDC induced killing of cells, which express CLD18A2 but    not of cells which express CLD18A1 (e.g. 26D12, 28D10, 37H8, and    39F11, 163E12, ch-125E1, ch-163E12, ch-175D10)-   e) mediating ADCC induced killing of cells expressing CLD18 (e.g.    26B5, 37G11, 37H8, 38G5, 38H3, 39F11, 43A11, 47D12, and 61C2,    ch-163E12, ch-175D10)-   f) mediating ADCC induced killing but not CDC mediated killing of    cells expressing CLD18 (e.g. 37G11, 42E12, and 43A11)-   g) mediating ADCC induced killing and CDC induced killing of cells    expressing CLD18A2 (e.g. 37H8, 38143, 39F11, ch-163E12, ch-175D10).

As exemplified herein, antibodies of the invention further encompassesmolecules, which

-   a) bind to differentiated cells of normal stomach, but not to stem    cells of stomach (e.g. 39F11)-   b) do not bind to normal gastric tissue as well as other normal    organs but exclusively to cancer cells (e.g. 26B5)-   c) bind to an epitope encompassing a non-glycosylated Asn at    position 116 of CLD18-   d) which bind to human as well as to mouse CLD18 allowing to    thoroughly perform preclinical toxicity studies in mice.

Antibodies of the invention may be derived from different species,including but not limited to mouse, rat, rabbit, guinea pig and human.Antibodies of the invention also include chimeric molecules in which anantibody constant region derived from one species, preferably human, iscombined with the antigen binding site derived from another species.Moreover antibodies of the invention include humanized molecules inwhich the antigen binding, sites of an antibody derived from a non-humanspecies are combined with constant and framework regions of humanorigin.

Antibodies of the invention include polyclonal and monoclonal antibodiesand include IgG2a (e.g. IgG2a, κ, λ), IgG2b (e.g. IgG2b, κ, λ), IgG3(e.g. IgG3, κ, λ) and IgM antibodies. However, other antibody isotypesare also encompassed by the invention, including IgG1, IgA1, IgA2,secretory IgA, IgD, and IgE antibodies. The antibodies can be wholeantibodies or antigen-binding fragments thereof including, for example,Fab, F(ab′)₂, Fv, single chain Fv fragments or bispecific antibodies.Furthermore, the antigen-binding fragments include binding-domainimmunoglobulin fusion proteins comprising (i) a binding domainpolypeptide (such as a heavy chain variable region or a light chainvariable region) that is fused to an immunoglobulin hinge regionpolypeptide, (ii) an immunoglobulin heavy chain CH2 constant regionfused to the hinge region, and (iii) an immunoglobulin heavy chain CH3constant region fused to the CH2 constant region. Such binding-domainimmunoglobulin fusion proteins are further disclosed in US2003/0118592and US 2003/0133939.

Antibodies of the present invention preferably dissociate from CLD18with a dissociation equilibrium constant (KD) of approximately 1-100 nMor less. Preferably, antibodies of the invention do not cross-react withrelated cell-surface antigens and thus do not inhibit their function.

In preferred embodiments, antibodies of the present invention can becharacterized by one or more of the following properties:

-   a) specificity for CLD18, in particular specificity for CLD18A2;-   b) a binding affinity to CLD18, in particular CLD18A2, of about 100    nM or less, preferably, about 5-10 nM or less and, more preferably,    about 1-3 nM or less,-   c) the ability to mediate a high level of CDC on either CD55/59    negative or CD55/59 positive, cells;-   d) the ability to inhibit the growth of cells which express CLD18;-   e) the ability to induce apoptosis of cells which express CLD18;-   f) the ability to induce homotypic adhesion of cells which express    CLD18;-   g) the ability to induce ADCC of cells which express CLD18 in the    presence of effector cells;-   h) the ability to prolong survival of a subject having tumor cells    which express CLD18;-   i) the ability to deplete cells which express CLD18;-   j) the ability to deplete cells which express low levels of CLD18    and/or-   k) the ability to aggregate CLD18 on the surface of living cells

The anti-CLD18 antibodies of the present invention can be derivatized,linked to or co-expressed to other binding specificities. In aparticular embodiment, the invention provides a bispecific ormultispecific molecule comprising at least one first binding specificityfor CLD18 (e.g., an anti-CLD18 antibody or mimetic thereof), and asecond binding specificity for a effector cell, such as a bindingspecificity for an Fc receptor (e.g., a Fc-gamma receptor, such asFc-gamma RI, or any other Fc receptor) or a T cell receptor, e.g., CD3.

Accordingly, the present invention includes bispecific and multispecificmolecules that bind to both CLD18 and to an Fc receptor or a T cellreceptor, e.g. CD3. Examples of Fc receptors are IgG receptor, Fc-gammareceptor (FcγR), such as FcγRI (CD64), FcγRII (CD32), and FcγRIII(CD16). Other Fc receptors, such as IgA receptors (e.g., FcαRI), alsocan be targeted. The Fc receptor is preferably located on the surface ofan effector cell, e.g., a monocyte, macrophage or an activatedmononuclear cell. In a preferred embodiment, the bispecific andmultispecific molecules bind to an Fc receptor at a site which is,distinct from the immunoglobulin Fc (e.g., IgG or IgA) binding site ofthe receptor. Therefore, the binding of the bispecific and multispecificmolecules is not blocked by physiological levels of immunoglobulins.

In yet another aspect, anti-CLD18 antibodies of the invention arederivatized, linked to or co-expressed with another functional molecule,e.g., another peptide or protein (e.g., a Fab′ fragment). For example,an antibody of the invention can be functionally linked (e.g., bychemical coupling, genetic fusion, noncovalent association or otherwise)to one or more other molecular entities, such as another antibody (e.g.to produce a bispecific or a multispecific antibody), a cytotoxin,cellular ligand or antigen (e.g. to produce an immunoconjugate, such asan immunotoxin). An antibody of the present invention can be linked toother therapeutic moieties, e.g., a radioisotope, a small moleculeanti-cancer drug, a recombinant cytokine or chemokine. Accordingly, thepresent invention encompasses a large variety of antibody conjugates,bispecific and multispecific molecules, and fusion proteins, all ofwhich bind to CLD18 expressing cells and which can be used to targetother molecules to such cells.

In still another aspect, the invention provides compositions, e.g.,pharmaceutical and diagnostic compositions/kits, comprising apharmaceutically acceptable carrier formulated along with one or acombination of antibodies of the invention. In a particular embodiment,the composition includes a combination of antibodies which bind todistinct epitopes or which possess distinct functional characteristics,such as inducing CDC and/or ADCC and inducing apoptosis. In thisembodiment of the invention, antibodies may be used in combination,e.g., as a pharmaceutical composition comprising two or more anti-CLD18monoclonal antibodies. For example, anti-CLD18 antibodies havingdifferent but complementary activities can be combined in a singletherapy to achieve a desired therapeutic effect. In a preferredembodiment, the composition includes an anti-CLD18 antibody thatmediates CDC combined with another anti-CLD18 antibody that inducesapoptosis. In another embodiment, the composition includes an anti-CLD18antibody that mediates highly effective killing of target cells in thepresence of effector cells, combined with another anti-CLD18 antibodythat inhibits the growth of cells expressing CLD18.

The present invention also includes the simultaneous or sequentialadministration of two or more anti-CLD18 antibodies of the invention,wherein at least one of said antibodies is a chimeric anti-CLD18antibody and at least one further antibody is a human anti-CLD18antibody, the antibodies binding to the same or different epitopes ofCLD18. Preferably, a chimeric CLD18 antibody of the invention isadministered first followed by the administration of a human anti-CLD18antibody of the invention, wherein the human anti-CLD18 antibody ispreferably administered for an extended period of time, i.e. asmaintenance therapy.

Antibodies, immunoconjugates, bispecific and multispecific molecules andcompositions of the present invention can be used in a variety ofmethods for inhibiting growth of cells expressing CLD18, in particularCLD18A2 and/or selectively killing cells expressing CLD18, in particularCLD18A2 by contacting the cells with an effective amount of theantibody, immunconjugate, bispecific/multispecific molecule orcomposition, such that the growth of the cell is inhibited and/or thecell is killed. In one embodiment, the method includes killing of thecell expressing CLD18, optionally in the presence of effector cells, forexample, by CDC, apoptosis, ADCC, phagocytosis, or by a combination oftwo or more of these mechanisms. Cells expressing CLD18 which can beinhibited or killed using the antibodies of the invention include cancercells such as tumorigenic stomach, pancreatic, esophageal, lung,ovarian, colon, hepatic, head-neck, and gallbladder cells.

Accordingly, antibodies of the present invention can be used to treatand/or prevent a variety of diseases involving cells expressing CLD18 byadministering the antibodies to patients suffering from such diseases.Exemplary diseases that can be treated (e.g., ameliorated) or preventedinclude, but are not limited to, tumorigenic diseases. Examples oftumorigenic diseases, which can be treated and/or prevented includegastric cancer, pancreatic cancer, esophageal cancer, lung cancer,ovarian cancer, colorectal cancer, hepatic cancer, head-neck cancer, andcancer of the gallbladder.

In a particular embodiment of the invention, the subject beingadministered the antibody is additionally treated with achemotherapeutic agent, radiation, or an agent that modulates, e.g.,enhances or inhibits, the expression or activity of an Fc receptor, e.g.an Fc-gamma receptor, such as a cytokine. Typical cytokines foradministration during treatment include granulocyte colony-stimulatingfactor (G-CSF), granulocyte-macrophage colony-stimulating factor(GM-CSF), interferon-γ (IFN-γ), and tumor necrosis factor (TNF). Typicaltherapeutic agents include, among others, anti-neoplastic agents such asdoxorubicin, cisplatin, taxotere, 5-fluoruracil, methotrexat, gemzitabinand cyclophosphamide.

In yet another aspect, the invention relates to an immunization strategyto immunize non-human animals such as mice with human CLD18 or a peptidefragment thereof, preferably CLD18A2 or a peptide fragment thereof toobtain antibodies. Preferred peptides for immunization are thoseselected from the group consisting of SEQ ID NO: 2, 4, 6, 16, 18, 20-23,and 26-31. Accordingly, in preferred embodiments, the antibodies of theinvention are those obtained by immunization using peptides selectedfrom the group consisting of SEQ ID NO: 2, 4, 6, 16, 18, 20-23, and26-31. Analogously, antibodies to CLD18 can be generated in a transgenicnon-human animal, such as a transgenic mouse. The transgenic non-humananimal may be a transgenic mouse having a genome comprising a heavychain transgene and a light chain transgene encoding all or a portion ofan antibody.

Wildtype as well as transgenic non-human animals can be immunized with apurified or enriched preparation of CLD18 antigen and/or nucleic acidsand/or to cells expressing CLD18 or a peptide fragment thereof.Preferably, the non-human animal, is capable of producing multipleisotypes of human monoclonal antibodies to CLD18 (e.g., IgG, IgA and/orIgM) by undergoing V-D-J recombination and isotype switching. Isotypeswitching may occur by e.g., classical or non-classical isotypeswitching.

Accordingly, in yet another aspect, the invention provides isolated Bcells from a non-human animal as described above. The isolated B cellscan then be immortalized by fusion to an immortalized cell to provide asource (e.g., a hybridoma) of antibodies of the invention. Suchhybridomas (i.e., which produce antibodies of the invention) are alsoincluded within the scope of the invention.

As exemplified herein, antibodies of the invention can be obtaineddirectly from hybridomas which express the antibody, or can be clonedand recombinantly expressed in a host cell (e.g., a CHO cell, or alymphocytic cell). Further examples of host cells are microorganisms,such as E. coli, and fungi, such as yeast. Alternatively, they can beproduced recombinantly in a transgenic non-human animal or plant.

Preferred hybridoma cells for producing antibodies of the invention arethose sequenced or deposited at the DSMZ (Mascheroder Weg 1b, 31824Braunschweig, Germany; new address: Inhoffenstr. 7B, 31824 Braunschweig,Germany) having the following designations and accession numbers:

-   a. 182-D1106-055 accession no. DSM ACC2737, deposited on Oct. 19,    2005-   b. 182-D1106-056, accession no. DSM ACC2738, deposited on Oct. 19,    2005-   c. 182-D1106-057, accession no. DSM ACC2739, deposited on Oct. 19,    2005-   d. 182-D1106-058, accession no. DSM ACC2740, deposited on Oct. 19,    2005-   e. 182-D1106-059, accession no. DSM ACC2741, deposited on Oct. 19,    2005-   f. 182-D1106-062, accession no. DSM ACC2742, deposited on Oct. 19,    2005,-   g. 182-D1106-067, accession no. DSM ACC2743, deposited on Oct. 19,    2005-   h. 182-D758-035, accession no. DSM ACC2745, deposited on Nov. 17,    2005-   i. 182-D758-036, accession no. DSM ACC2746, deposited on Nov. 17,    2005-   j. 182-D758-040, accession no. DSM ACC2747, deposited on Nov. 17,    2005-   k. 182-D1106-061, accession no. DSM ACC2748, deposited on Nov. 17,    2005-   l. 182-D1106-279, accession no. DSM ACC2808, deposited on Oct. 26,    2006-   m. 182-D1106-294, accession no. DSM ACC2809, deposited on Oct. 26,    2006,-   n. 182-D1106-362, accession no. DSM ACC2810, deposited on Oct. 26,    2006.

Preferred antibodies of the invention are those produced by andobtainable from the above-described hybridomas; i.e. 37G11 in the caseof 182-D1106-055, 37H8 in the case of 182-D1106-056, 38G5 in the case of182-D1106-057, 38H3 in the case of 182-D1106-058, 39F11 in the case of182-D1106-059, 43A11 in the case of 182-D1106-062, 61C2 in the case of182-D1106-067, 26B5 in the case of 182-D758-035, 26D12 in the case of182-D758-036, 28D10 in the case of 182-D758-040, 42E12 in the case of182-D1106-061, 125E1 in the case of 182-D1106-279, 163E12 in the case of182-D1106-294, and 175D10 in the case of 182-D1106-362; and thechimerized and humanized forms thereof.

In preferred embodiments, antibodies, in particular chimerised forms ofantibodies according to the invention include antibodies comprising aheavy chain constant region (CH) comprising an amino acid sequencederived from a human heavy chain constant region such as the amino acidsequence represented by SEQ ID NO: 46 or 150 or a fragment thereof. Infurther preferred embodiments, antibodies, in particular chimerisedforms of antibodies according to the invention include antibodiescomprising a light chain constant region (CL) comprising an amino acidsequence derived from a human light chain constant region such as theamino acid sequence represented by SEQ ID NO: 41 or 148 or a fragmentthereof. In a particular preferred embodiment, antibodies, in particularchimerised forms of antibodies according to the invention includeantibodies which comprise a CH comprising an amino acid sequence derivedfrom a human CH such as the amino acid sequence represented by SEQ IDNO: 46 or 150 or a fragment thereof and which comprise a CL comprisingan amino acid sequence derived from a human CL such as the amino acidsequence represented by SEQ ID NO: 41 or 148 or a fragment thereof.

A CH comprising the amino acid sequence represented by SEQ ID NO: 46 maybe encoded by a nucleic acid comprising the nucleic acid sequencerepresented by SEQ ID NO: 45. A CH comprising the amino acid sequencerepresented by SEQ ID NO: 150 may be encoded by a nucleic acidcomprising the nucleic acid sequence represented by SEQ ID NO: 149. A CLcomprising the amino acid sequence represented by SEQ ID NO: 41 may beencoded by a nucleic acid comprising the nucleic acid sequencerepresented by SEQ ID NO: 40. A CL comprising the amino acid sequencerepresented by SEQ ID NO: 148 may be encoded by a nucleic acidcomprising the nucleic acid sequence represented by SEQ ID NO: 147.

In certain preferred embodiments, chimerised forms of antibodies includeantibodies comprising a heavy chain comprising an amino acid sequenceselected from the group consisting of SEQ ID NO: 115, 116, 117, 118,119, 120, and a fragment thereof and/or comprising a light chaincomprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 121, 122, 123, 124, 125, 126, 127, 128, 129, and a fragmentthereof.

In certain preferred embodiments, chimerised forms of antibodies includeantibodies comprising a combination of heavy chains and light chainsselected from the following possibilities (i) to (ix):

(i) the heavy chain comprises an amino acid sequence represented by SEQID NO: 115 or a fragment thereof and the light chain comprises an aminoacid sequence represented by SEQ ID NO 122 or a fragment thereof,

(ii) the heavy chain comprises an amino acid sequence represented by SEQID NO: 116 or a fragment thereof and the light chain comprises an aminoacid sequence represented by SEQ ID NO: 121 or a fragment thereof,

(iii) the heavy chain comprises an amino acid sequence represented bySEQ ID NO: 117 or a fragment thereof and the light chain comprises anamino acid sequence represented by SEQ ID NO: 123 or a fragment thereof,

(iv) the heavy chain comprises an amino acid sequence represented by SEQID NO: 119 or a fragment thereof and the light chain comprises an aminoacid sequence represented by SEQ ID NO: 126 or a fragment thereof,

(v) the heavy chain comprises an amino acid sequence represented by SEQID NO: 118 or a fragment thereof and the light chain comprises an aminoacid sequence represented by SEQ ID NO: 125 or a fragment thereof,

(vi) the heavy chain comprises an amino acid sequence represented by SEQID NO: 120 or a fragment thereof and the light chain comprises an aminoacid sequence represented by SEQ ID NO: 124 or a fragment thereof,

(vii) the heavy chain comprises an amino acid sequence represented bySEQ ID NO: 120 or a fragment thereof and the light chain comprises anamino acid sequence represented by SEQ ID NO: 127 or a fragment thereof,

(viii) the heavy chain comprises an amino acid sequence represented bySEQ ID NO: 120 or a fragment thereof and the light chain comprises anamino acid sequence represented by SEQ ID NO: 128 or a fragment thereof,and

(ix) the heavy chain comprises an amino acid sequence represented by SEQID NO: 120 or a fragment thereof and the light chain comprises an aminoacid sequence represented by SEQ ID NO: 129 or a fragment thereof.

“Fragment” or “fragment of an amino acid sequence” as used above relatesto a part of an antibody sequence, i.e. a sequence which represents theantibody sequence shortened at the N- and/or C-terminus, which when itreplaces said antibody sequence in an antibody retains binding of saidantibody to CLD18 and preferably functions of said antibody as describedherein, e.g. CDC mediated lysis or ADCC mediated lysis. Preferably, afragment of an amino acid sequence comprises at least 80%, preferably atleast 90%, 95%, 96%, 97%, 98%, or 99% of the amino acid residues fromsaid amino acid sequence. A fragment of an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 115, 116, 117, 118, 119, 120,121, 122, 123, 124, 125; 126, 127, 128, and 129 preferably relates tosaid sequence wherein 17, 18, 19, 20, 21, 22 or 23 amino acids at theN-terminus are removed. Fragments of amino acid sequences describedherein may be encoded by respective fragments of nucleic acid sequencesencoding said amino acid sequences.

A heavy chain comprising an amino acid sequence represented by SEQ IDNO: 115 may be encoded by a nucleic acid comprising the nucleic acidsequence represented by SEQ ID NO: 100. A heavy chain comprising anamino acid sequence represented by SEQ ID NO: 116 may be encoded by anucleic acid comprising the nucleic acid sequence represented by SEQ IDNO: 101. A heavy chain comprising an amino acid sequence represented bySEQ ID NO: 117 may be encoded by a nucleic acid comprising the nucleicacid sequence represented by SEQ ID NO: 102. A heavy chain comprising anamino acid sequence represented by SEQ ID NO: 119 may be encoded by anucleic acid comprising the nucleic acid sequence represented by SEQ IDNO: 104. A heavy chain comprising an amino acid sequence represented bySEQ ID NO: 118 may be encoded by a nucleic acid comprising the nucleicacid sequence represented by SEQ ID NO: 103. A heavy chain comprising anamino acid sequence represented by SEQ ID NO: 120 may be encoded by anucleic acid comprising the nucleic acid sequence represented by SEQ IDNO: 105.

A light chain comprising an amino acid sequence represented by SEQ IDNO: 122 may be encoded by a nucleic acid comprising the nucleic acidsequence represented by SEQ ID NO: 107. A light chain comprising anamino acid sequence represented by SEQ. ID NO: 121 may be encoded by anucleic acid comprising the nucleic acid sequence represented by SEQ IDNO: 106. A light chain comprising an amino acid sequence represented bySEQ ID NO: 123 may be encoded by a nucleic acid comprising the nucleicacid sequence represented by SEQ ID NO: 108. A light chain comprising anamino acid sequence represented by SEQ ID NO: 126 may be encoded by anucleic acid comprising the nucleic acid sequence represented by SEQ IDNO: 111. A light chain comprising an amino acid sequence represented bySEQ ID NO: 125 may be encoded by a nucleic acid comprising the nucleicacid sequence represented by SEQ ID NO: 110. A light chain comprising anamino acid sequence represented by SEQ ID NO: 124 may be encoded by anucleic acid comprising the nucleic acid sequence represented by SEQ IDNO: 109. A light chain comprising an amino acid sequence represented bySEQ ID NO: 127 may be encoded by a nucleic acid comprising the nucleicacid sequence represented by SEQ ID NO: 112. A light chain comprising anamino acid sequence represented by SEQ ID NO: 128 may be encoded by anucleic acid comprising the nucleic acid sequence represented by SEQ IDNO: 113. A light chain comprising an amino acid sequence represented bySEQ ID NO: 129 may be encoded by a nucleic acid comprising the nucleicacid sequence represented by SEQ ID NO: 114.

In a preferred embodiment, an antibody of the invention comprises aheavy chain variable region (VH) comprising an amino acid sequenceselected from the group consisting of SEQ ID NO: 132, 133, 134, 135,136, 137, and a fragment thereof.

In a preferred embodiment, an antibody of the invention comprises alight chain variable region (VL) comprising an amino acid sequenceselected from the group consisting of SEQ ID NO: 138, 139, 140, 141,142, 143, 144, 145, 146, and a fragment thereof.

In certain preferred embodiments, an antibody of the invention comprisesa combination of heavy chain variable region (VII) and light chainvariable region (VL) selected from the following possibilities (i) to(ix):

(i) the VH comprises an amino acid sequence represented by SEQ ID NO:132 or a fragment thereof and the VL comprises an amino acid sequencerepresented by SEQ ID NO: 139 or a fragment thereof,

(ii) the VH comprises an amino acid sequence represented by SEQ ID NO:133 or a fragment thereof and the VL comprises an amino acid sequencerepresented by SEQ ID NO: 138 or a fragment thereof,

(iii) the VH comprises an amino acid sequence represented by SEQ ID NO:134 or a fragment thereof and the VL comprises an amino acid sequencerepresented by SEQ ID NO: 140 or a fragment thereof,

(iv) the VH comprises an amino acid sequence represented by SEQ ID NO:136 or a fragment thereof and the VL comprises an amino acid sequencerepresented by SEQ ID NO: 143 or a fragment thereof,

(v) the VH comprises an amino acid sequence represented by SEQ ID NO:135 or a fragment thereof and the VL comprises an amino acid sequencerepresented by SEQ ID NO: 142 or a fragment thereof,

(vi) the VH comprises an amino acid sequence represented by SEQ ID NO:137 or a fragment thereof and the VL comprises an amino acid sequencerepresented by SEQ ID NO: 141 or a fragment thereof,

(vii) the VH comprises an amino acid sequence represented by SEQ ID NO:137 or a fragment thereof and the VL comprises an amino acid sequencerepresented by SEQ ID NO: 144 or a fragment thereof,

(viii) the VH comprises an amino acid sequence represented by SEQ ID NO:137 or a fragment thereof and the VL comprises an amino acid sequencerepresented by SEQ ID NO: 145 or a fragment thereof,

(ix) the VH comprises an amino acid sequence represented by SEQ ID NO:137 or a fragment thereof and the VL comprises an amino acid sequencerepresented by SEQ ID NO: or a fragment thereof.

A VH comprising an amino acid sequence represented by SEQ ID NO: 132 maybe encoded by a nucleic acid comprising the nucleic acid sequencerepresented by SEQ ID NO: 55. A VH comprising an amino acid sequencerepresented by SEQ ID NO: 13.3 may be encoded by a nucleic acidcomprising the nucleic acid sequence represented by SEQ ID NO: 56. A VHcomprising an amino acid sequence represented by SEQ ID NO: 134 may beencoded, by a nucleic acid comprising the nucleic acid sequencerepresented by SEQ ID NO: 57. A VH comprising an amino acid sequencerepresented by SEQ ID NO: 136 may be encoded by a nucleic acidcomprising the nucleic acid sequence represented by SEQ ID NO: 59. A VIIcomprising an amino acid sequence represented by SEQ ID NO: 135 may beencoded by a nucleic acid comprising the nucleic acid sequencerepresented by SEQ ID NO: 58. A VII comprising an amino acid sequencerepresented by SEQ ID NO: 137 may be encoded by a nucleic acidcomprising the nucleic acid sequence represented by SEQ ID NO: 60.

A VL comprising an amino acid sequence represented by SEQ ID NO: 139 maybe encoded by a nucleic acid comprising the nucleic acid sequencerepresented by SEQ ID NO: 62. A VL comprising an amino acid sequencerepresented by SEQ ID NO: 138 may be encoded by a nucleic acidcomprising the nucleic acid sequence represented by SEQ ID NO: 61. A VLcomprising an amino acid sequence represented by SEQ ID NO: 140 may beencoded by a nucleic acid comprising the nucleic acid sequencerepresented by SEQ ID NO: 63. A VL comprising an amino acid sequencerepresented by SEQ ID NO: 143 may be encoded by a nucleic acidcomprising the nucleic acid sequence represented by SEQ ID NO: 66. A VLcomprising an amino acid sequence represented by SEQ ID NO: 142 may beencoded by a nucleic acid comprising the nucleic acid sequencerepresented by SEQ ID NO: 65. A VL comprising an amino acid sequencerepresented by SEQ ID NO: 141 may be encoded by a nucleic acidcomprising the nucleic acid sequence represented by SEQ ID NO: 64. A VLcomprising an amino acid sequence represented by SEQ ID NO: 144 may beencoded by a nucleic acid comprising the nucleic acid sequencerepresented by SEQ ID NO: 67. A VL comprising an amino acid sequencerepresented by SEQ ID NO: 145 may be encoded by a nucleic acidcomprising the nucleic acid sequence represented by SEQ ID NO: 68. A VLcomprising an amino acid sequence represented by SEQ ID NO: 146 may beencoded by a nucleic acid comprising the nucleic acid sequencerepresented by SEQ ID NO: 69.

In a preferred embodiment, an antibody of the invention comprises a VHcomprising a set of complementarity-determining regions CDR1, CDR2 andCDR3 selected from the following embodiments (i) to (vi):

(i) CDR1: positions 45-52 of SEQ ID NO: 115, CDR2: positions 70-77 ofSEQ ID NO: 115, CDR3: positions 116-125 of SEQ ID NO: 115,

(ii) CDR1: positions 45-52 of SEQ ID NO: 116, CDR2: positions 70-77 ofSEQ ID NO: 116, CDR3: positions 116-126 of SEQ ID NO: 116,

(iii) CDR1: positions 45-52 of SEQ ID NO: 117, CDR2: positions 70-77 ofSEQ ID NO: 117, CDR3: positions 116-124 of SEQ ID NO: 117,

(iv) CDR1: positions 45-52 of SEQ ID NO: 118, CDR2: positions 70-77 ofSEQ ID NO: 118, CDR3: positions 116-126 of SEQ ID NO: 118,

(v) CDR1: positions 44-51 of SEQ ID NO: 119, CDR2: positions 69-76 ofSEQ ID NO: 119, CDR3: positions 115-125 of SEQ ID NO: 119, and

(vi) CDR1: positions 45-53 of SEQ ID NO: 120, CDR2: positions 71-78 ofSEQ ID NO: 120, CDR3: positions 117-128 of SEQ ID NO: 120.

In a preferred embodiment, an antibody of the invention comprises a VLcomprising a set of complementarity-determining regions CDR1, CDR2 andCDR3 selected from the following embodiments (i) to (ix):

(i) CDR1: positions 47-58 of SEQ ID NO: 121, CDR2: positions 76-78 ofSEQ ID NO: 121, CDR3: positions 115-123 of SEQ ID NO: 121,

(ii) CDR1: positions 49-53 of SEQ ID NO: 122, CDR2: positions 71-73 ofSEQ ID NO: 122, CDR3: positions 110-118 of SEQ ID NO: 122,

(iii) CDR1: positions 47-52 of SEQ ID NO: 123, CDR2: positions 70-72 ofSEQ ID NO: 123, CDR3: positions 109-117 of SEQ ID NO: 123,

(iv) (iv) CDR1: positions 47-58 of SEQ ID NO: 124, CDR2: positions 76-78of SEQ ID NO: 124, CDR3: positions 115-123 of SEQ ID NO: 124,

(v) CDR1: positions 47-58 of SEQ ID NO: 125, CDR2: positions 76-78 ofSEQ ID NO: 125, CDR3: positions 115-123 of SEQ ID NO: 125,

(vi) CDR1: positions 47-58 of SEQ ID NO: 126, CDR2: positions 76-78 ofSEQ ID NO: 126, CDR3: positions 115-122 of SEQ ID NO: 126,

(vii) CDR1: positions 47-58 of SEQ ID NO: 127, CDR2: positions 76-78 ofSEQ ID NO: 127, CDR3: positions 115-123 of SEQ ID NO: 127,

(viii) CDR1: positions 47-58 of SEQ ID NO: 128, CDR2: positions 76-78 ofSEQ ID NO: 128, CDR3: positions 115-123 of SEQ ID NO: 128, and

(ix) CDR1: positions 47-52 of SEQ ID NO: 129, CDR2: positions 70-72 ofSEQ ID NO: 129, CDR3: positions 109-117 of SEQ ID NO: 129.

In a preferred embodiment, an antibody of the invention comprises acombination of VH and VL each comprising a set ofcomplementarity-determining regions CDR1, CDR2 and CDR3 selected fromthe following embodiments (i) to (ix):

(i) VH: CDR1: positions 45-52 of SEQ ID NO: 115, CDR2: positions 70-77of SEQ ID NO: 115, CDR3: positions 116-125 of SEQ ID NO: 115, VL: CDR1:positions 49-53 of SEQ ID NO: 122, CDR2: positions 71-73 of SEQ ID NO:122, CDR3: positions 110-118 of SEQ ID NO: 122,(ii) VH: CDR1: positions 45-52 of SEQ ID NO: 116, CDR2: positions 70-77of SEQ ID NO: 116, CDR3: positions 116-126 of SEQ ID NO: 116, VL: CDR1:positions 47-58 of SEQ ID NO: 121, CDR2: positions 76-78 of SEQ ID NO:121, CDR3: positions 115-123 of SEQ ID NO: 121,(iii) VH: CDR1: positions 45-52 of SEQ ID NO: 117, CDR2: positions 70-77of SEQ ID NO: 117, CDR3: positions 116-124 of SEQ ID NO: 117, VL: CDR1:positions 47-52 of SEQ ID NO: 123, CDR2: positions 70-72 of SEQ ID NO:123, CDR3: positions 109-117 of SEQ ID NO: 123,(iv) VH: CDR1: positions 44-51 of SEQ ID NO: 119, CDR2: positions 69-76of SEQ ID NO: 119, CDR3: positions 115-125 of SEQ ID NO: 119, VL: CDR1:positions 47-58 of SEQ ID NO: 126, CDR2: positions 76-78 of SEQ ID NO:126, CDR3: positions 115-122 of SEQ ID NO: 126,(v) VH: CDR1: positions 45-52 of SEQ ID NO: 118, CDR2: positions/70-77of SEQ ID NO: 118, CDR3: positions 116-126 of SEQ ID NO: 118, VL: CDR1:positions 47-58 of SEQ ID NO: 125, CDR2: positions 76-78 of SEQ ID NO:125, CDR3: positions 115-123 of SEQ ID NO: 125,(vi) VH: CDR1: positions 45-53 of SEQ ID NO: 120, CDR2: positions 71-78of SEQ ID NO: 120, CDR3: positions 117-128 of SEQ ID NO: 120, VL: CDR1:positions 47-58 of SEQ ID NO: 124, CDR2: positions 76-78 of SEQ ID NO:124, CDR3: positions 115-123 of SEQ ID NO: 124,(vii) VH: CDR1: positions 45-53 of SEQ ID NO: 120, CDR2: positions 71-78of SEQ ID NO: 120, CDR3: positions 117-128 of SEQ ID NO: 120, VL: CDR1:positions 47-58 of SEQ ID NO: 127, CDR2: positions 76-78 of SEQ ID NO:127, CDR3: positions 115-123 of SEQ ID NO: 127,(viii) VH:CDR1: positions 45-53 of SEQ ID NO: 120, CDR2: positions 71-78of SEQ ID NO: 120, CDR3: positions 117-128 of SEQ ID NO: 120, VL: CDR1:positions 47-58 of SEQ ID NO: 128, CDR2: positions 76-78 of SEQ ID NO:128, CDR3: positions 115-123 of SEQ ID NO: 128, and(ix) VH: CDR1: positions 45-53 of SEQ ID NO: 120, CDR2: positions 71-78of SEQ ID NO: 120, CDR3: positions 117-128 of SEQ ID NO: 120, VL: CDR1:positions 47-52 of SEQ ID NO: 129, CDR2: positions 70-72 of SEQ ID NO:129, CDR3: positions 109-117 of SEQ ID NO: 129.

In further preferred embodiments, an antibody of the inventionpreferably comprises one or more of the complementarity-determiningregions (CDRs), preferably at least the CDR3 variable region, of theheavy chain variable region (VH) and/or of the light chain variableregion (VL) of a monoclonal antibody against CLD18, preferably of amonoclonal antibody against CLD18 described herein, and preferablycomprises one or more of the complementarity-determining regions (CDRs),preferably at least the CDR3 variable region, of the heavy chainvariable regions (VH) and/or light chain variable regions (VL) describedherein. In one embodiment said one or more of thecomplementarity-determining regions (CDRs) are selected from a set ofcomplementarity-determining regions CDRI, CDR2 and CDR3 describedherein. In a particularly preferred embodiment, an antibody of theinvention preferably comprises the complementarity-determining regionsCDR1, CDR2 and CDR3 of the heavy chain variable region (VH) and/or ofthe light chain variable region (VL) of a monoclonal antibody againstCLD18, preferably of a monoclonal antibody against CLD18 describedherein, and preferably comprises the complementarity-determining regionsCDR1, CDR2 and CDR3 of the heavy chain variable regions (VH) and/orlight chain variable regions (VL) described herein.

In one embodiment an antibody of the invention comprising one or moreCDRs, a set of CDRs or a combination of sets of CDRs as described hereincomprises said CDRs together with their intervening framework regions.Preferably, the portion will also include at least about 50% of eitheror both of the first and fourth framework regions, the 50% being theC-terminal 50% of the first framework region and the N-terminal 50% ofthe fourth framework region. Construction of antibodies of the presentinvention made by recombinant DNA techniques may result in theintroduction of residues N- or C-terminal to the variable regionsencoded by linkers introduced to facilitate cloning or othermanipulation steps, including the introduction of linkers to joinvariable regions of the invention to further protein sequences includingimmunoglobulin heavy chains, other variable domains (for example in theproduction of diabodies) or protein labels.

In one embodiment an antibody of the invention comprising one or moreCDRs, a set of CDRs or a combination of sets of CDRs as described hereincomprises said CDRs in a human antibody framework.

Reference herein to an antibody comprising with respect to the heavychain thereof a particular chain, or a particular region or sequencepreferably relates to the situation wherein all heavy chains of saidantibody comprise said particular chain, region or sequence. Thisapplies correspondingly to the light chain of an antibody.

The present invention also relates to nucleic acids comprising genes ornucleic acid sequences encoding antibodies or parts thereof, e.g. anantibody chain, as described herein. The nucleic acids may be comprisedin a vector, e.g., a plasmid, cosmid, virus, bacteriophage or anothervector used e.g. conventionally in genetic engineering. The vector maycomprise further genes such as marker genes which allow for theselection of the vector in a suitable host cell and under suitableconditions. Furthermore, the vector may comprise expression controlelements allowing proper expression of the coding regions in suitablehosts. Such control elements are known to the artisan and may include apromoter, a splice cassette; and a translation initiation codon.

Preferably, the nucleic acid of the invention is operatively attached tothe above expression control sequences allowing expression in eukaryoticor prokaryotic cells. Control elements ensuring expression in eukaryoticor prokaryotic cells are well known to those skilled in the art.

Methods for construction of nucleic acid molecules according to thepresent invention, for construction of vectors comprising the abovenucleic acid molecules, for introduction of the vectors intoappropriately chosen host cells, for causing or achieving the expressionare well-known in the art.

A further aspect of the present invention relates to a host cellcomprising a nucleic acid or vector as disclosed herein.

Other features and advantages of the instant invention will be apparentfrom the following detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an immunfluorescence: analysis of HEK293 cells transfectedwith CLD18A2 coupled to a green fluorochrome and reacted with mouseserum after DNA immunisation with SEQ ID NO: 15 fused to a helperepitope.

FIG. 2 shows a Western blot analysis of HEK293 cells transfected withCLD18A2-myc (SEQ ID NO: 3) and untransfected HEK293 cells with themonoclonal mouse-anti-c-myc antibody 9E11 (Serotec, CRL MCA2200).

FIG. 3 shows an immunfluorescence analysis using CHO cells transfectedwith CLD18A2 and a polyclonal rabbit-anti-CLD18 antibody (Zymed, CRL38-8000).

FIGS. 4A and B show the binding of hybridoma supernatants 24H5 and 85A3to HEK293 cells transiently transfected with human CLD18A2 and afluorescent marker as determined by flow cytometry. FIG. 4C shows thebinding of hybridoma supernatants 45C1, 125E1, 163E12, 166E2 and 175D10to HEK293 cells stably transfected with human CLD18A2 and counterstainedwith propidium iodide.

FIG. 5 shows binding of hybridoma supernatants 24H5 (A), 9E8 (B), 26B5(C) and 19B9 (D) to HEK293 cells transiently transfected with afluorescent marker and either human CLD18A2 or CLD18A2-Myc or CLD18A2-HAas analyzed by flow cytometry.

FIGS. 6A and B show binding of hybridoma supernatants 37H8, 43A11, 45C1and 163E12 to HEK293 cells stably transfected with either human CLD18A2or CLD18A1 as determined by flow cytometry.

FIG. 7 shows an immunfluorescence analysis of the CLD18A2 isoformspecific monoclonal antibody 37G11 by staining HEK293 cells transfectedwith CLD18A2 (A, C) and CLD18A1 (B, D), respectively, under native (A,B) and paraformaldehyde fixation (C, D) conditions.

FIG. 8 shows an immunfluorescence analysis of the CLD18 monoclonalantibody 26B5 by staining HEK293 cells transfected with CLD18A2 (A, C)and CLD18A1 (B, D), respectively, under native (A, B) andparaformaldehyde fixation (C, D) conditions.

FIG. 9. Cell line RT-PCR

RT-PCR analysis with CLD18A2-specific primers showed clear expression in4/5 tested cell lines.

FIG. 10 shows an immunfluorescence analysis of DAN-G cells (subclone F2)and a polyclonal rabbit-anti-CLD18 antibody (Zymed, CRL 38-8000).

FIG. 11 shows an immunfluorescence analysis of KATO-III cells (subclone3B9 4D5) and a polyclonal rabbit-anti-CLD18 antibody (Zymed, CRL38-8000).

FIG. 12 A shows an immunfluorescence analysis of SNU-16 cells (subcloneG5) with a polyclonal rabbit-anti-CLD18 antibody (Zymed, CRL 38-8000).FIG. 12 B shows an immunfluorescence analysis of KATO-III cells withmonoclonal antibodies of the invention.

FIG. 13 shows surface expression of CLD18 on KATO-III and NUGC-4 cellsas analyzed by staining of cells with monoclonal antibodies 61C2 and163E12 followed by flow cytometrical analysis.

FIG. 14. Protein-alignment of human CLD18A1 (NP_057453), human CLD18A2(NP_001002026), mouse CLD18A1 (NP_062789) and mouse CLD18A2 (AAL15636).

FIGS. 15 A and B show binding of hybridoma supernatants 38G5, 38H3,37G11, 45C1, and 163E12, respectively, to HEK293 cells transientlytransfected with a fluorescent marker and either murine CLD18A1 ormurine CLD18A2 as analyzed by flow cytometry.

FIG. 16. Immunohistochemical analyses with polyclonal AB p105.Immunohistochemical stainings on a subset of normal tissues (stomach,lung, bone marrow and prostate) confirm gastric tissue specificity (A).Expression was also detected in stomach carcinomas (upper row) and lungcarcinomas (B). Only differentiated cells but not stem cells do expressCLD18A2 (C).

FIG. 17. Immunohistochemical analyses with monoclonal AB 39F11D7

(A) Specific protein expression was detected in normal stomach mucosa,whereas all other tested normal tissue were negative.

(B) Strong CLD18A2 expression was found in stomach and lung carcinomas.

FIG. 18. Immunohistochemical analyses with monoclonal AB 26B5 (A),175D10 (B), 43A11 (C), 163E12 (D), and 45C1 (E). All antibodies showstrong staining of HEK293-CLD18A2 xenograft tumors and gastric cancerspecimens, but not HEK293-Mock control-transfected tumors.

FIG. 19 is a graph comparing the percentage of dead cells afterinduction of CDC by 85A3, 28D10, 24H5, or 26D12 against HEK293 cellsstably transfected with human CLD18A2 using flow cytometry.

FIG. 20 is a graph comparing the percentage of specific cell lysis afterinduction of CDC by 24H5, 26D12, 28D10, 37G11, 37H8, 38G5, 38H3, 39F11,4106, 42E12, 43A11, 44E10, 47D12, or 61C2 against adherent CHO cellsstably transfected with either human CLD18A2 or human CLD18A1 asdetermined by fluorescence measurement.

FIG. 21 shows concentration-dependent induction of CDC against CHO cellsstably transfected with human CLD18A2 by 75B8 (A), 28D10 (B), or 37H8(C) as determined by fluorescence measurement.

FIG. 22 shows lysis of HEK293-CLD18A2 cells by 26B5, 37H8, 38G5, 47D12,and 61C2, respectively, in the presence of MNCs.

FIG. 23 shows lysis of HEK293-CLD18A1 cells by 26B5, 37H8, 38G5, 47D12,and 61C2, respectively, in the presence of MNCs.

FIG. 24 shows tumor growth inhibition by antibodies of the invention inan early treatment xenograft model with HEK293-CLD18A2 cells.

FIGS. 25A and B show prolonged survival by treatment with antibodies ofthe invention in two early treatment xenograft models withHEK293-CLD18A2 cells.

FIG. 26 shows prolongation of survival by antibodies of the invention inan advanced treatment xenograft model with HEK293-CLD18A2 cells.

FIG. 27A shows tumor growth inhibition by antibodies of the invention inan early treatment xenograft model. FIG. 27B shows prolongation ofsurvival by antibodies of the invention in an early treatment xenograftmodel. Endogenously CLD18A2 expressing DAN-G cells were used.

FIG. 28 shows CLD18A2 mRNA expression in mouse tissues. RT-PCRinvestigations with CLD18A2-specific primers showed no significantexpression within all tested normal tissues except stomach. Thefollowing normal tissues were analysed: 1: small intestine, 2: spleen,3: skin, 4: stomach, 5: lung, 6: pancreas, 7: lymph node, 8: thymus, 9:negative control

FIG. 29 shows CLD18 expression in normal stomach. Immunohistochemicalanalysis with CLD18 specific antibody of mouse stomach reveals conservedexpression pattern. While the surface epithelia and deeper cryptsexpress CLD18 in their cell surface, the central neck region is CLD18negative.

FIG. 30 shows haematoxylin and eosin staining of mice stomach tissues.Shown is in overview (A) and in detail (B) the stomach of a37G11-treated mouse in comparison to a control mouse (C and D), whichwas treated with PBS only.

FIGS. 31A and B show flowcytometric staining of HEK293 cells stablytransfected with human CLD18A1 and A2, respectively, as well asendogenously expressing KATO-III cells with antibodies of the invention(43A11, 125E1, 163E12, 166E2, and 175D10).

FIG. 32 shows CDC on CLD18A2 expressing cells mediated by chimericantibodies of the invention.

FIG. 33 shows ADCC on KATO-III cells mediated by chimeric antibodies ofthe invention.

DETAILED DESCRIPTION OF THE INVENTION

The antibodies described herein may be isolated monoclonal antibodieswhich specifically bind to an epitope present on CLD18. Isolatedmonoclonal antibodies encompassed by the present invention include IgA,IgG1-4, IgE, IgM, and IgD antibodies. In one embodiment the antibody isan IgG1 antibody, more particularly an IgG1, kappa or IgG1, lambdaisotype. In another embodiment the antibody is an IgG3 antibody, moreparticularly an IgG3, kappa or IgG3, lambda isotype. In yet anotherembodiment the antibody is an IgG4 antibody, more particularly an IgG4,kappa or IgG4, lambda isotype. In still another embodiment the antibodyis an IgA1 or IgA2 antibody. In still another embodiment the antibody isan IgM antibody.

In one embodiment the invention relates to antibodies which specificallybind to cells expressing CLD18, and preferably (i) bind to cellsexpressing CLD18A2, and (ii) do not bind to cells not expressing CLD18A2but expressing CLD18A1. The antibodies of the invention preferably (i)mediate killing of cells expressing CLD18A2, and (ii) do not mediatekilling of cells not expressing CLD18A2 but expressing CLD18A1.

In another embodiment, the invention relates to antibodies which (i)bind to tumor cells expressing CLD18, (ii) do not bind to CLD18expressing cells of normal stomach mucosa, and/or (iii) do not bind toCLD18 expressing cells of non-cancer lung tissue.

The invention also includes antibodies which (i) mediate killing oftumor cells expressing CLD18, (ii) do not mediate killing of CLD18expressing cells of normal stomach mucosa, and/or (iii) do not mediatekilling of CLD18 expressing cells of non-cancer lung tissue.

In particular embodiments, the antibodies of the invention (i) bind toan epitope on CLD18A2 which is not present on CLD18A1, preferably SEQ IDNO: 21, 22, and 23, (ii) bind to an epitope localized on theCLD18A2-loop1, preferably SEQ ID NO: 28, (iii) bind to an epitopelocalized on the CLD18A2-loop2, preferably SEQ ID NO: 30, (iv) bind toan epitope localized on the CLD18A2-loopD3, preferably SEQ ID NO: 31,(v) bind to an epitope, which encompass CLD18A2-loop1 andCLD18A2-loopD3, (vi) bind to a non-glycosylated epitope localized on theCLD18A2-loopD3, preferably SEQ ID NO: 29, or (vii) bind to an epitopepresent in human and mouse CLD18 (SEQ ID NO: 2, SEQ ID NO: 8 and SEQ IDNO: 35, SEQ ID NO: 37, respectively).

In particularly preferred embodiments, the antibodies of the inventionbind to an epitope on CLD18A2 which is not present on CLD18A1.

Antibodies of the invention include fully human antibodies. Suchantibodies may be produced in a non-human transgenic animal, e.g., atransgenic mouse, capable of producing multiple isotypes of humanmonoclonal antibodies to CLD18 by undergoing V-D-J recombination andisotype switching. Such, transgenic animal can also be a transgenicrabbit for producing polyclonal antibodies such as disclosed in US2003/0017534.

Binding of an antibody of the invention to the CLD18 antigen may mediatethe killing of cells expressing CLD18 (e.g. a tumor cell), e.g. byactivation of the complement system. The killing of cells expressingCLD18 may occur by one or more of the following mechanisms: complementdependent cytotoxity (CDC) of cells expressing CLD18; apoptosis of cellsexpressing CLD18; effector cell phagocytosis of cells expressing CLD18;or effector cell antibody dependent cellular cytotoxicity (ADCC) ofcells expressing CLD18.

In order that the present invention may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

DEFINITION OF TERMS

The term “CLD18” relates to claudin-18 and includes any variants,including CLD18A1 and CLD18A2, conformations, isoforms and specieshomologs of CLD18 which are naturally expressed by cells or areexpressed by cells transfected with the CLD18 gene. Preferably, “CLD18”relates to human CLD18, in particular CLD18A2 (SEQ ID NOs: 1, 2) and/orCLD18A1 (SEQ ID NOs: 7, 8), more preferably CLD18A2.

The term “CLD18A1” includes posttranslationally modified variants,isoforms and species homologs of human CLD18A1 which are naturallyexpressed by cells or are expressed on cells transfected with theCLD18A1 gene.

The term “CLD18A2” includes posttranslationally modified variants,isoforms and species homologs of human CLD18A2 which are naturallyexpressed by cells or are expressed on cells transfected with theCLD18A2 gene.

The term “CLD18 variant” shall encompass (i) CLD18 splice variants, (ii)CLD18-posttranslationally modified variants, particularly includingvariants with different N-glycosylation status, (iii) CLD18 conformationvariants, particularly including CLD18-conformation-1,CLD18-conformation-2 and CLD18-conformation-3, (iv) CLD18 free andhomotypically/heterotypically associated variants localized atintercellular tight junctions, (v) CLD18 cancer related and CLD18non-cancer related variants.

The term “raft” refers to the sphingolipid- and cholesterol-richmembrane microdomains located in the outer leaflet area of the plasmamembrane of a cell. The ability of certain proteins to associate withinsuch domains and their ability of forming “aggregates” or “focalaggregates” can effect the protein's function. For example, thetranslocation of CLD18 molecules into such structures, after being boundby antibodies of the present invention, creates a high density of CLD18antigen-antibody complexes in the plasma membranes. Such a high densityof CLD18 antigen-antibody complexes can enable efficient activation ofthe complement system during CDC.

The terms “conformation” and “topology” describe how an integralmembrane molecule is positioned in the cell surface membrane, and, inparticular, which of its regions are extracellular and thus eligible forantibodies. CLD18 for example can exist in three differentconformations, which most likely depend on whether it is prevalent ashomomers or heteromers and whether it is integrated in supramoleculartight junction structures or “free”. These different states result indifferent epitopes eligible to antibodies.

According to the invention, the term “disease” refers to anypathological state, including cancer, in particular those forms ofcancer described herein.

By “tumor” is meant an abnormal group of cells or tissue that grows by arapid, uncontrolled cellular proliferation and continues to grow afterthe stimuli that initiated the new growth cease. Tumors show partial orcomplete lack of structural organization and functional coordinationwith the normal tissue, and usually form a distinct mass of tissue,which may be either benign or malignant.

By “metastasis” is meant the spread of cancer cells from its originalsite to another part of the body. The formation of metastasis is a verycomplex process and depends on detachment of malignant cells from theprimary tumor, invasion of the extracellular matrix, penetration of theendothelial basement membranes to enter the body cavity and vessels, andthen, after being transported by the blood, infiltration of targetorgans. Finally, the growth of a new tumor at the target site depends onangiogenesis. Tumor metastasis often occurs even after the removal ofthe primary tumor because tumor cells or components may remain anddevelop metastatic potential. In one embodiment, the “metastasis”according to the invention relates to “distant metastasis” which relatesto a metastasis which is remote from the primary tumor and the regionallymph node system.

The term “treatment of a disease” includes curing, shortening theduration, ameliorating, preventing, slowing down or inhibitingprogression or worsening, or preventing or delaying the onset of adisease or the symptoms thereof.

According to the invention, a sample may be any sample useful accordingto the present invention, in particular a biological sample such atissue sample, including bodily fluids, and/or a cellular sample and maybe obtained in the conventional manner such as by tissue biopsy,including punch biopsy, and by taking blood, bronchial aspirate, sputum,urine, feces or other body fluids. According to the invention, the term“biological sample” also includes fractions of biological samples.

The term “antibody” refers to a glycoprotein comprising at least twoheavy (H) chains and two light (L) chains inter-connected by disulfidebonds, or an antigen binding portion thereof. The term “antibody” alsoincludes all recombinant forms of antibodies, in particular of theantibodies described herein, e.g., antibodies expressed in prokaryotes,unglycosylated antibodies, and any antigen-binding antibody fragmentsand derivatives as described below. Each heavy chain is comprised of aheavy chain variable region (abbreviated herein as VH) and a heavy chainconstant region. Each light chain is comprised of a light chain variableregion (abbreviated herein as VL) and a light chain constant region. TheVH and VL regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR). Each VH and VL is composed of three CDRs and four FRs,arranged from amino-terminus to carboxy-terminus in the following order:FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavyand light chains contain a binding domain that interacts with anantigen. The constant regions of the antibodies may mediate the bindingof the immunoglobulin to host tissues or factors, including variouscells of the immune system (e.g., effector cells) and the firstcomponent (C1q) of the classical complement system.

The term “humanized antibody” refers to a molecule having an antigenbinding site that is substantially derived from an immunoglobulin from anon-human species, wherein the remaining immunoglobulin structure of themolecule is based upon the structure and/or sequence of a humanimmunoglobulin. The antigen binding site may either comprise completevariable domains fused onto constant domains or only the complementaritydetermining regions (CDR) grafted onto appropriate framework regions inthe variable domains. Antigen binding sites may be wild-type or modifiedby one or more amino acid substitutions, e.g. modified to resemble humanimmunoglobulins more closely. Some forms of humanized antibodiespreserve all CDR sequences (for example a humanized mouse antibody whichcontains all six CDRs from the mouse antibody). Other forms have one ormore CDRs which are altered with respect to the original antibody.

The term “chimeric antibody” refers to those antibodies wherein oneportion of each of the amino acid sequences of heavy and light chains ishomologous to corresponding sequences in antibodies derived from aparticular species or belonging to a particular class, while theremaining segment of the chain is homologous to corresponding sequencesin another. Typically the variable region of both light and heavy chainsmimics the variable regions of antibodies derived from one species ofmammals, while the constant portions are homologous to sequences ofantibodies derived from another. One clear advantage to such chimericforms is that the variable region can conveniently be derived frompresently known sources using readily available B-cells or hybridomasfrom non-human host organisms in combination with constant regionsderived from, for example, human cell preparations. While the variableregion has the advantage of ease of preparation and the specificity isnot affected by the source, the constant region being human, is lesslikely to elicit an immune response from a human subject when theantibodies are injected than would the constant region from a non humansource. However the definition is not limited to this particularexample.

The term “antigen-binding portion” of an antibody (or simply “bindingportion”), as used herein, refers to one or more fragments of anantibody that retain the ability to specifically bind to an antigen. Ithas been shown that the antigen-binding function of an antibody can beperformed by fragments of a full-length antibody. Examples of bindingfragments encompassed within the term “antigen binding portion” of anantibody include (i) Fab fragments, monovalent fragments consisting ofthe VL, VH, CL and CH domains; (ii) F(ab′)₂ fragments, bivalentfragments comprising two Fab fragments linked by a disulfide bridge atthe hinge region; (iii) Fd fragments consisting of the VH and CHdomains; (iv) Fv fragments consisting of the VL and VH domains of asingle arm of an antibody, (v) dAb fragments (Ward et al., (1989) Nature341: 544-546), which consist of a VH domain; (vi) isolatedcomplementarity determining regions (CDR), and (vii) combinations of twoor more isolated CDRs which may optionally be joined by a syntheticlinker. Furthermore, although the two domains of the Fv fragment, VL andVH, are coded for by separate genes, they can be joined, usingrecombinant methods, by a synthetic linker that enables them to be madeas a single protein chain in which the VL and VH regions pair to formmonovalent molecules (known as single chain Fv (scFv); see e.g., Bird etal. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl.Acad. Sci. USA 85: 5879-5883). Such single chain antibodies are alsointended to be encompassed within the term “antigen-binding portion” ofan antibody. A further example is binding-domain immunoglobulin fusionproteins comprising (i) a binding domain polypeptide that is fused to animmunoglobulin hinge region polypeptide, (ii) an immunoglobulin heavychain CH2 constant region fused to the hinge region, and (iii) animmunoglobulin heavy chain CH3 constant region fused to the CH2 constantregion. The binding domain polypeptide can be a heavy chain variableregion or a light chain variable region. The binding-domainimmunoglobulin fusion proteins are further disclosed in US 2003/0118592and US 2003/0133939. These antibody fragments are obtained usingconventional techniques known to those with skill in the art, and thefragments are screened for utility in the same manner as are intactantibodies.

The term “epitope” means a protein determinant capable of binding to anantibody, wherein the term “binding” herein preferably relates to aspecific binding. Epitopes usually consist of chemically active surfacegroupings of molecules such as amino acids or sugar side chains andusually have specific three dimensional structural characteristics, aswell as specific charge characteristics. Conformational andnon-conformational epitopes are distinguished in that the binding to theformer but not the latter is lost in the presence of denaturingsolvents.

The term “discontinuous epitope” as used herein, means a conformationalepitope on a protein antigen which is formed from at least two separateregions in the primary sequence of the protein.

The term “bispecific molecule” is intended to include any agent, e.g., aprotein, peptide, or protein or peptide complex, which has two differentbinding specificities. For example, the molecule may bind to, orinteract with (a) a cell surface antigen, and (b) an Fc receptor on thesurface of an effector cell. The term “multispecific molecule” or“heterospecific molecule” is intended to include any agent, e.g., aprotein, peptide, or protein or peptide complex, which has more than twodifferent binding specificities. For example, the molecule may bind to,or interact with (a) a cell surface antigen, (b) an Fc receptor on thesurface of an effector cell, and (c) at least one other component.Accordingly, the invention includes, but is not limited to, bispecific,trispecific, tetraspecific, and other multispecific molecules which aredirected to CLD18, and to other targets, such as Fc receptors oneffector cells. The term “bispecific antibodies” also includesdiabodies. Diabodies are bivalent, bispecific antibodies in which the VHand VL domains are expressed on a single polypeptide chain, but using alinker that is too short to allow for pairing between the two domains onthe same chain, thereby forcing the domains to pair with complementarydomains of another chain and creating two antigen binding sites (seee.g., Holliger, P., et at. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure 2: 1121-1123).

The invention also includes derivatives of the antibodies describedherein. The term “antibody derivatives” refers to any modified form ofan antibody, e.g., a conjugate of the antibody and another agent orantibody. As used herein, an antibody is “derived from” a particulargermline sequence if the antibody is obtained from a system byimmunizing an animal or by screening an immunoglobulin gene library, andwherein the selected antibody is at least 90%, more preferably at least95%, even more preferably at least 96%, 97%, 98%, or 99% identical inamino acid sequence to the amino acid sequence encoded by the germlineimmunoglobulin gene. Typically, an antibody derived from a particulargermline sequence will display no more than 10 amino acid differences,more preferably, no more than 5, or even more preferably, no more than4, 3, 2, or 1 amino acid difference from the amino acid sequence encodedby the germline immunoglobulin gene.

As used herein, the term “heteroantibodies” refers to two or moreantibodies, derivatives thereof, or antigen binding regions linkedtogether, at least two of which have different specificities. Thesedifferent specificities include a binding specificity for an Fc receptoron an effector cell, and a binding specificity for an antigen or epitopeon a target cell, e.g., a tumor cell.

The antibodies described herein may be human antibodies. The term “humanantibody”, as used herein, is intended to include antibodies havingvariable and constant regions derived from human germline immunoglobulinsequences. The human antibodies of the invention may include amino acidresidues not encoded by human germline immunoglobulin sequences (e.g.,mutations introduced by random or site-specific mutagenesis in vitro orby somatic mutation in vivo).

The term “monoclonal antibody” as used herein refers to a preparation ofantibody molecules of single molecular composition. A monoclonalantibody displays a single binding specificity and affinity for aparticular epitope. In one embodiment, the monoclonal antibodies areproduced by a hybridoma which includes a B cell obtained from anon-human animal, e.g., mouse, fused to an immortalized cell.

The term “recombinant antibody”, as used herein, includes all antibodiesthat are prepared, expressed, created or isolated by recombinant means,such as (a) antibodies isolated from an animal (e.g., a mouse) that istransgenic or transchromosomal with respect to the immunoglobulin genesor a hybridoma prepared therefrom, (b). antibodies isolated from a hostcell transformed to express the antibody, e.g., from a transfectoma, (c)antibodies isolated from a recombinant, combinatorial antibody library,and (d) antibodies prepared, expressed, created or isolated by any othermeans that involve splicing of immunoglobulin gene sequences to otherDNA sequences.

The term “transfectoma”, as used herein, includes recombinant eukaryotichost cells expressing an antibody, such as CHO cells, NS/0 cells, HEK293cells, HEK293T cells, plant cells, or fungi, including yeast cells.

As used herein, a “heterologous antibody” is defined in relation to atransgenic organism producing such an antibody. This term refers to anantibody having an amino acid sequence or an encoding nucleic acidsequence corresponding to that found in an organism not consisting ofthe transgenic organism, and being generally derived from a speciesother than the transgenic organism.

As used herein, a “heterohybrid antibody” refers to an antibody havinglight and heavy chains of different organismal origins. For example, anantibody having a human heavy chain associated with a murine light chainis a heterohybrid antibody.

The antibodies described herein are preferably isolated. An “isolatedantibody” as used herein, is intended to refer to an antibody which issubstantially free of other antibodies having different antigenicspecificities (e.g., an isolated antibody that specifically binds toCLD18 is substantially free of antibodies that specifically bindantigens other than CLD18). An isolated antibody that specifically bindsto an epitope, isoform or variant of human CLD18 may, however, havecross-reactivity to other related antigens, e.g., from other species(e.g., CLD18 species homologs). Moreover, an isolated antibody may besubstantially free of other cellular material and/or chemicals. In oneembodiment of the invention, a combination of “isolated” monoclonalantibodies relates to antibodies having different specificities andbeing combined in a well defined composition.

According to the invention, the term “binding” preferably relates to“specific binding”. As used herein, “specific binding” refers toantibody binding to a predetermined antigen. Typically, the antibodybinds with an affinity corresponding to a KD of about 1×10⁻⁷ M or less,and binds to the predetermined antigen with an affinity corresponding toa KD that is at least two orders of magnitude lower than its affinityfor binding to a non-specific antigen (e.g., BSA, casein) other than thepredetermined antigen or a closely-related antigen.

The term “KD” (M), as used herein, is intended to refer to thedissociation equilibrium constant of a particular antibody-antigeninteraction.

As used herein, “isotype” refers to the antibody class (e.g., IgM orIgG1) that is encoded by heavy chain constant region genes.

As used herein, “isotype switching” refers to the phenomenon by whichthe class, or isotype, of an antibody changes from one Ig class to oneof the other Ig classes.

The term “naturally occurring” as used herein as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory isnaturally occurring.

The term “rearranged” as used herein refers to a configuration of aheavy chain or light chain immunoglobulin locus wherein a V segment ispositioned immediately adjacent to a D-J or J segment in a conformationencoding essentially a complete VH or VL domain, respectively. Arearranged immunoglobulin (antibody) gene locus can be identified bycomparison to germline DNA; a rearranged locus will have at least onerecombined heptamer/nonamer homology element.

The term “unrearranged” or “germline configuration” as used herein inreference to a V segment refers to the configuration wherein the Vsegment is not recombined so as to be immediately adjacent to a D or Jsegment.

The teen “nucleic acid molecule”, as used herein, is intended to includeDNA molecules and RNA molecules. A nucleic acid molecule may besingle-stranded or double-stranded, but preferably is double-strandedDNA.

The nucleic acids described according to the invention have preferablybeen isolated. The term “isolated nucleic acid” means according to theinvention that the nucleic acid was (i) amplified in vitro, for exampleby polymerase chain reaction (PCR), (ii) recombinantly produced bycloning, (iii) purified, for example by cleavage and gel-electrophoreticfractionation, or (iv) synthesized, for example by chemical synthesis.An isolated nucleic acid is a nucleic acid which is available formanipulation by recombinant DNA techniques.

Nucleic acids may, according to the invention, be present alone or incombination with other nucleic acids, which may be homologous orheterologous. In preferred embodiments, a nucleic acid is functionallylinked to expression control sequences which may be homologous orheterologous with respect to said nucleic acid. The term “homologous”means that a nucleic acid is also functionally linked to the expressioncontrol sequence naturally and the term “heterologous” means that anucleic acid is not functionally linked to the expression controlsequence naturally.

A. nucleic acid, such as a nucleic acid expressing RNA and/or protein orpeptide, and an expression control sequence are “functionally” linked toone another, if they are covalently linked to one another in such a waythat expression or transcription of said nucleic acid is under thecontrol or under the influence of said expression control sequence. Ifthe nucleic acid is to be translated into a functional protein, then,with an expression control sequence functionally linked to a codingsequence, induction of said expression control sequence results intranscription of said nucleic acid, without causing a frame shift in thecoding sequence or said coding sequence not being capable of beingtranslated into the desired protein or peptide.

The term “expression control sequence” comprises according to theinvention promoters, ribosome binding sites, enhancers and other controlelements which regulate transcription of a gene or translation of amRNA. In particular embodiments of the invention, the expression controlsequences can be regulated. The exact structure of expression controlsequences may vary as a function of the species or cell type, butgenerally comprises 5′-untranscribed and 5′- and 3′, untranslatedsequences which are involved in initiation of transcription andtranslation, respectively, such as TATA box, capping sequence, CAATsequence, and the like. More specifically, 5′-untranscribed expressioncontrol sequences comprise a promoter region which includes a promotersequence for transcriptional control of the functionally linked nucleicacid. Expression control sequences may also comprise enhancer sequencesor upstream activator sequences.

According to the invention the term “promoter” or “promoter region”relates to a nucleic acid sequence which is located upstream (5′) to thenucleic acid sequence being expressed and controls expression of thesequence by providing a recognition and binding site for RNA-polymerase.The “promoter region” may include further recognition and binding sitesfor further factors which are involved in the regulation oftranscription of a gene. A promoter may control the transcription of aprokaryotic or eukaryotic gene. Furthermore, a promoter may be“inducible” and may initiate transcription in response to an inducingagent or may be “constitutive” if transcription is not controlled by aninducing agent. A gene which is under the control of an induciblepromoter is not expressed or only expressed to a small extent if aninducing agent is absent. In the presence of the inducing agent the geneis switched on or the level of transcription is increased. This ismediated, in general, by binding of a specific transcription factor.

Promoters which are preferred according to the invention includepromoters for SP6, T3 and T7 polymerase, human U6 RNA promoter, CMVpromoter, and artificial hybrid promoters thereof (e.g. CMV) where apart or parts are fused to a part or parts of promoters of genes ofother cellular proteins such as e.g. human GAPDH(glyceraldehyde-3-phosphate dehydrogenase), and including or notincluding (an) additional intron(s).

According to the invention, the term “expression” is used in its mostgeneral meaning and comprises the production of RNA or of RNA andprotein/peptide. It also comprises partial expression of nucleic acids.Furthermore, expression may be carried out transiently or stably.

In a preferred embodiment, a nucleic acid molecule is according to theinvention present in a vector, where appropriate with a promoter, whichcontrols expression of the nucleic acid. The term “vector” is used herein its most general meaning and comprises any intermediary vehicle for anucleic acid which enables said nucleic acid, for example, to beintroduced into prokaryotic and/or eukaryotic cells and, whereappropriate, to be integrated into a genome. Vectors of this kind arepreferably replicated and/or expressed in the cells. Vectors compriseplasmids, phagemids, bacteriophages or viral genomes. The term “plasmid”as used herein generally relates to a construct of extrachromosomalgenetic material, usually a circular DNA duplex, which can replicateindependently of chromosomal DNA.

As the vector for expression of an antibody, either of a vector type inwhich the antibody heavy chain and light chain are present in differentvectors or a vector type in which the heavy chain and light chain arepresent in the same vector can be used.

The teaching given herein with respect to specific nucleic acid andamino acid sequences, e.g. those shown in the sequence listing, is to beconstrued so as to also relate to modifications of said specificsequences resulting in sequences which are functionally equivalent tosaid specific sequences, e.g. amino acid sequences exhibiting propertiesidentical or similar to those of the specific amino acid sequences andnucleic acid sequences encoding amino acid sequences exhibitingproperties identical or similar to those of the amino acid sequencesencoded by the specific nucleic acid sequences. One important propertyis to retain binding of an antibody to its target or to sustain effectorfunctions of an antibody. Preferably, a sequence modified with respectto a specific sequence, when it replaces the specific sequence in anantibody retains binding of said antibody to CLD18 and preferablyfunctions of said antibody as described herein; e.g. CDC mediated lysisor ADCC mediated lysis.

It will be appreciated by those skilled in the art that in particularthe sequences of the CDR, hypervariable and variable regions can bemodified without losing the ability to bind CLD18. For example, CDRregions will be either identical or highly homologous to the regionsspecified herein. By “highly homologous” it is contemplated that from 1to 5, preferably from 1 to 4, such as 1 to 3 or 1 or 2 substitutions maybe made in the CDRs. In addition, the hypervariable and variable regionsmay be modified so that they show substantial homology with the regionsspecifically disclosed herein.

It is to be understood that the specific nucleic acids described hereinalso include nucleic acids modified for the sake of optimizing the codonusage in a particular host cell or organism. Differences in codon usageamong organisms can lead to a variety of problems concerningheterologous gene expression. Codon optimization by changing one or morenucleotides of the original sequence can result in an optimization ofthe expression of a nucleic acid, in particular in optimization oftranslation efficacy, in a homologous or heterologous host in which saidnucleic acid is to be expressed. For example if nucleic acids derivedfrom human and encoding constant regions and/or framework regions ofantibodies are to be used according to the present invention, e.g. forpreparing chimeric or humanized antibodies, it may be preferred tomodify said nucleic acids for the sake of optimization of codon usage,in particular if said nucleic acids, optionally fused to heterologousnucleic acids such as nucleic acids derived from other organisms asdescribed herein, are to be expressed in cells from an organismdifferent from human such as mouse or hamster. For example, the nucleicacid sequences encoding human light and heavy chain constant regionssuch as those according to SEQ ID NOs: 40 and 45, respectively, can bemodified to include one or more, preferably, at least 1, 2, 3, 4, 5, 10,15, 20 and preferably up to 10, 15, 20, 25, 30, 50, 70 or 100 or morenucleotide replacements resulting in an optimized codon usage but notresulting in a change of the amino acid sequence. Such nucleotidereplacements preferably relate to replacements of nucleotides in SEQ IDNos: 40 and 45, respectively, selected from the replacements shown inthe following alignment of SEQ ID Nos: 40 and 45, respectively, withtheir modified counterparts and not resulting in a change in the encodedamino acid sequence or relate to corresponding replacements atcorresponding positions in other nucleic acid sequences encoding humanlight and heavy chain constant regions, respectively. Preferably, all ofthe replacements shown in the following alignments of SEQ ID Nos: 40 and45, respectively, with their modified counterparts not resulting in achange in the encoded amino acid sequence are effected in nucleic acidsequences encoding human light and heavy chain constant regions,respectively.

Furthermore, it may be desired according to the present invention tomodify the amino acid sequences described herein, in particular those ofhuman heavy chain constant regions to adapt the sequence to a desiredallotype, e.g. an allotype found in the Caucasian population. Suchmodifications are preferably selected from the group consisting of thefollowing amino acid replacements within SEQ ID NO: 46 or atcorresponding positions within other human heavy chain constant regions:K93R, D235E, and L237M. Preferably, all of these modifications areincluded in amino acid sequences of human heavy chain constant regions.

According the invention, the term “corresponding positions” relates tonucleotides or amino acid residues which in a sequence alignment of twonucleic acid or protein sequences are aligned to each other.

Preferably the degree of identity between a specific nucleic acidsequence described herein and a nucleic acid sequence which is modifiedwith respect to said specific nucleic acid sequence will be at least70%, preferably at least 75%, more preferably at least 80%, even morepreferably at least 90% or most preferably at least 95%, 96%, 97%, 98%or 99%. Preferably, the two sequences are capable of hybridizing andforming a stable duplex with one another, with hybridization preferablybeing carried out under conditions which allow specific hybridizationbetween polynucleotides (stringent conditions). Stringent conditions aredescribed, for example, in Molecular Cloning: A Laboratory Manual, J.Sambrook et al., Editors, 2nd Edition, Cold Spring Harbor Laboratorypress, Cold Spring Harbor, N.Y.; 1989 or Current Protocols in MolecularBiology, F. M. Ausubel et al., Editors, John Wiley & Sons, Inc., NewYork and refer, for example, to hybridization at 65° C. in hybridizationbuffer (3.5×SSC, 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovineserum albumin, 2.5 mM NaH₂PO₄ (pH 7), 0.5% SDS, 2 mM EDTA). SSC is 0.15M sodium chloride/0.15 M sodium citrate, pH 7. After hybridization, themembrane to which the DNA has been transferred is washed, for example,in 2×SSC at room temperature and then in 0.1-0.5×SSC/0.1×SDS attemperatures of up to 68° C.

Preferably the degree of similarity, preferably identity between aspecific amino acid sequence described herein and an amino acid sequencewhich is modified with respect to said specific amino acid sequence suchas between amino acid sequences showing substantial homology will be atleast 70%, preferably at least 80%, even more preferably at least 90% ormost preferably at least 95%, 96%, 97%, 98% or 99%.

All of the above described modified sequences are within the scope ofthe present invention.

“Sequence similarity” indicates the percentage of amino acids thateither are identical or that represent conservative amino acidsubstitutions. “Sequence identity” between two polypeptide or nucleicacid sequences indicates the percentage of amino acids or nucleotidesthat are identical between the sequences.

The “percentage identity” is obtained after the best alignment, thispercentage being purely statistical and the differences between the twosequences being distributed randomly and over their entire length.Sequence comparisons between two nucleotide or amino acid sequences areconventionally carried out by comparing these sequences after havingaligned them optimally, said comparison being carried out by segment orby “window of comparison” in order to identify and compare local regionsof sequence similarity. The optimal alignment of the sequences forcomparison may be produced, besides manually, by means of the localhomology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482,by means of the local homology algorithm of Neddleman and Wunsch, 1970,J. Mol. Biol. 48, 443, by means of the similarity search method ofPearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85, 2444, or bymeans of computer programs which use these algorithms (GAP, BESTFIT,FASTA, BLAST P, BLAST N and TFASTA in Wisconsin Genetics SoftwarePackage, Genetics Computer Group, 575 Science Drive, Madison, Wis.).

The percentage identity is calculated by determining the number ofidentical positions between the two sequences being compared, dividingthis number by the number of positions compared and multiplying theresult obtained by 100 so as to obtain the percentage identity betweenthese two sequences.

“Conservative substitutions,” may be made, for instance, on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues involved.For example: (a) nonpolar (hydrophobic) amino acids include alanine,leucine, isoleucine, valine, proline, phenylalanine, tryptophan, andmethionine; (b) polar neutral amino acids include glycine, serine,threonine, cysteine, tyrosine, asparagine, and glutamine; (c) positivelycharged (basic) amino acids include arginine, lysine, and histidine; and(d) negatively charged (acidic) amino acids include aspartic acid andglutamic acid. Substitutions typically may be made within groups(a)-(d). In addition, glycine and proline may be substituted for oneanother based on their ability to disrupt [alpha]-helices. Somepreferred substitutions may be made among the following groups: (i) Sand T; (ii) P and G; and (iii) A, V, L and I. Given the known geneticcode, and recombinant and synthetic DNA techniques, the skilledscientist readily can construct DNAs encoding the conservative aminoacid variants.

The present invention comprises antibodies in which alterations havebeen made in the Fc region in order to change the functional orpharmacokinetic properties of the antibodies. Such alterations mayresult in a decrease or increase of C1q binding and CDC or of FcγRbinding and ADCC. Substitutions can, for example, be made in one or moreof the amino acid residues of the heavy chain constant region, therebycausing an alteration in an effector function while retaining theability to bind to the antigen as compared with the modified antibody,cf. U.S. Pat. No. 5,624,821 and U.S. Pat. No. 5,648,260.

The in vivo half-life of antibodies can be improved by modifying thesalvage receptor epitope of the Ig constant domain or an Ig-likeconstant domain such that the molecule does not comprise an intact CH2domain or an intact Ig Fc region, cf. U.S. Pat. No. 6,121,022 and U.S.Pat. No. 6,194,551. The in vivo half-life can furthermore be increasedby making mutations in the Fc region, e.g., by substituting threoninefor leucine at position 252, by substituting threonine for serine atposition 254, or by substituting threonine for phenylalanine at position256, cf. U.S. Pat. No. 6,277,375.

Furthermore, the glycosylation pattern of antibodies can be modified inorder to change the effector function of the antibodies. For example,the antibodies can be expressed in a transfectoma which does not add thefucose unit normally attached to Asn at position 297 of the Fc region inorder to enhance the affinity of the Fc region for Fc-Receptors which,in turn, will result in an increased ADCC of the antibodies in thepresence of NK cells, cf. Shield et al. (2002) JBC, 277: 26733.Furthermore, modification of galactosylation can be made in order tomodify CDC.

Alternatively, in another embodiment, mutations can be introducedrandomly along all or part of a anti-CLD18 antibody coding sequence,such as by saturation mutagenesis, and the resulting modified anti-CLD18antibodies can be screened for binding activity.

The term “recombinant host cell” (or simply “host cell”), as usedherein, is intended to refer to a cell into which a recombinantexpression vector has been introduced. It should be understood that suchterms are intended to refer not only to the particular subject cell butto the progeny of such a cell. Because certain modifications may occurin succeeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term “host cell” asused herein. Recombinant host cells include, for example, transfectomas,such as CHO cells, NS/0 cells, and lymphocytic cells.

As used herein, the term “subject” includes any human or non-humananimal. The term “non-human animal” includes all vertebrates, e.g.,mammals and non-mammals, such as non-human primates, sheep, dog, cow,chickens, amphibians, reptiles, etc.

The terms “transgenic animal” refers to an animal having a genomecomprising one or more transgenes, preferably heavy and/or light chaintransgenes, or transchromosomes (either integrated or non-integratedinto the animal's natural genomic DNA) and which is preferably capableof expressing the transgenes. For example, a transgenic mouse can have ahuman light chain transgene and either a human heavy chain transgene orhuman heavy chain transchromosome, such that the mouse produces humananti-CLD18 antibodies when immunized with CLD18 antigen and/or, cellsexpressing CLD18. The human heavy chain transgene can be integrated intothe chromosomal DNA of the mouse, as is the case for transgenic mice,e.g., HuMAb mice, such as HCo7 or HCo12 mice, or the human heavy chaintransgene can be maintained extrachromosomally, as is the case fortranschromosomal (e.g., KM) mice as described in WO 02/43478. Suchtransgenic and transchromosomal mice may be capable of producingmultiple isotypes of human monoclonal antibodies to CLD18 (e.g., IgG,IgA and/or IgE) by undergoing. V-D-J recombination and isotypeswitching.

Mechanisms of mAb Action

Although the following provides considerations regarding the mechanismunderlying the therapeutic efficacy of antibodies of the invention it isnot to be considered as limiting to the invention in any way.

The antibodies described herein preferably interact with components ofthe immune system, preferably through ADCC or CDC. Antibodies of theinvention can also be used to target payloads (e.g., radioisotopes,drugs or toxins) to directly kill tumor cells or can be usedsynergistically with traditional chemotherapeutic agents, attackingtumors through complementary mechanisms of action that may includeanti-tumor immune responses that may have been compromised owing to achemotherapeutic's cytotoxic side effects on T lymphocytes.

Antibody-Dependent Cell-Mediated Cytotoxicity.

ADCC describes the cell-killing ability of effector cells as describedherein, in particular lymphocytes, which preferably requires the targetcell being marked by an antibody.

ADCC preferably occurs when antibodies bind to antigens on tumor cellsand the antibody Fc domains engage Fc receptors (FcR) on the surface ofimmune effector cells. Several families of Fc receptors have beenidentified, and specific cell populations characteristically expressdefined Fc receptors. ADCC can be viewed as a mechanism to directlyinduce a variable degree of immediate tumor destruction that leads toantigen presentation and the induction of tumor-directed T-cellresponses. Preferably, in vivo induction of ADCC will lead totumor-directed T-cell responses and host-derived antibody responses.

Complement-Dependent Cytotoxicity.

CDC is another cell-killing method that can be directed by antibodies.IgM is the most effective isotype for complement activation. IgG1 andIgG3 are also both very effective at directing CDC via the classicalcomplement-activation pathway. Preferably, in this cascade, theformation of antigen-antibody complexes results in the uncloaking ofmultiple C1q binding sites in close proximity on the C_(H)2 domains ofparticipating antibody molecules such as IgG molecules (C1q is one ofthree subcomponents of complement C1). Preferably these uncloaked C1qbinding sites convert the previously low-affinity C1q-IgG interaction toone of high avidity, which triggers a cascade of events involving aseries of other complement proteins and leads to the proteolytic releaseof the effector-cell chemotactic/activating agents C3a and C5a.Preferably, the complement cascade ends in the formation of a membraneattack complex, which creates pores in the cell membrane that facilitatefree passage of water and solutes into and out of the cell.

Production of Antibodies

Antibodies of the invention can be produced by a variety of techniques,including conventional monoclonal antibody methodology, e.g., thestandard somatic cell hybridization technique of Kohler and Milstein,Nature 256: 495 (1975). Although somatic cell hybridization proceduresare preferred, in principle, other techniques for producing monoclonalantibodies can be employed, e.g., viral or oncogenic transformation ofB-lymphocytes or phage display techniques using libraries of antibodygenes.

The preferred animal system for preparing hybridomas that secretemonoclonal antibodies is the murine system. Hybridoma production in themouse is a very well established procedure. Immunization protocols andtechniques for isolation of immunized splenocytes for fusion are knownin the art. Fusion partners (e.g., murine myeloma cells) and fusionprocedures are also known.

Other preferred animal systems for preparing hybridomas that secretemonoclonal antibodies are the rat and the rabbit system (e.g. describedin Spieker-Polet et al., Proc. Natl. Acad. Sci. U.S.A. 92:9348 (1995),see also Rossi et al., Am. J. Clin. Pathol. 124: 295 (2005)).

In yet another preferred embodiment, human monoclonal antibodiesdirected against CLD18 can be generated using transgenic ortranschromosomal mice carrying parts of the human immune system ratherthan the mouse system. These transgenic and transchromosomic miceinclude mice known as HuMAb mice and KM mice, respectively, and arecollectively referred to herein as “transgenic mice.” The production ofhuman antibodies in such transgenic mice can be performed as describedin detail for CD20 in WO2004 035607.

Yet another strategy for generating monoclonal antibodies is to directlyisolate genes encoding antibodies from lymphocytes producing antibodiesof defined strategy e.g. see Babcock et al., 1996; A novel strategy forgenerating monoclonal antibodies from single, isolated lymphocytesproducing antibodies of defined strategy. For details of recombinantantibody engineering see also Welschof and Kraus, Recombinant antibodiesfor cancer therapy ISBN-0-89603-918-8 and Benny K. C. Lo AntibodyEngineering ISBN 1-58829-092-1.

Immunizations

To generate antibodies to CLD18, mice can be immunized withcarrier-conjugated peptides derived from the CLD18 sequence, an enrichedpreparation of recombinantly expressed CLD18 antigen or fragmentsthereof and/or cells expressing CLD18, as described: Alternatively, micecan be immunized with DNA encoding full length human CLD18 (e.g. SEQ IDNO: 1) or fragments thereof, in particular those of SEQ ID Nos:15, 17,and 19. In the event that immunizations using a purified or enrichedpreparation of the CLD18 antigen do not result in antibodies, mice canalso be immunized with cells expressing CLD18, e.g., a cell line, topromote immune responses.

The immune response can be monitored over the course of the immunizationprotocol with plasma and serum samples, being obtained by tail vein orretroorbital bleeds. Mice with sufficient titers of anti-CLD18immunoglobulin can be used for fusions. Mice can be boostedintraperitonealy or intravenously with CLD18 expressing cells 3 daysbefore sacrifice and removal of the spleen to increase the rate ofspecific antibody secreting hybridomas.

Generation of Hybridomas Producing Monoclonal Antibodies

To generate hybridomas producing monoclonal antibodies to CLD18,splenocytes and lymph node cells from immunized mice can be isolated andfused to an appropriate immortalized cell line, such as a mouse myelomacell line. The resulting hybridomas can then be screened for theproduction of antigen-specific antibodies. Individual wells can then bescreened by ELISA for antibody secreting hybridomas. ByImmunofluorescence and FACS analysis using CLD18 expressing cells,antibodies with specificity for CLD18 can be identified. The antibodysecreting hybridomas can be replated, screened again, and if stillpositive for anti-CLD18 monoclonal antibodies can be subcloned bylimiting dilution. The stable subclones can then be cultured in vitro togenerate antibody in tissue culture medium for characterization.

Generation of Transfectomas Producing Monoclonal Antibodies

Antibodies of the invention also can be produced in a host celltransfectoma using, for example, a combination of recombinant DNAtechniques and gene transfection methods as are well known in the art(Morrison, S. (1985) Science 229: 1202).

For example, in one embodiment, the gene(s) of interest, e.g., antibodygenes, can be ligated into an expression vector such as a eukaryoticexpression plasmid such as used by the GS gene expression systemdisclosed in WO 87/04462, WO 89/01036 and EP 338 841 or other expressionsystems well known in the art. The purified plasmid with the clonedantibody genes can be introduced in eukaryotic host cells such as CHOcells, NS/0 cells, HEK293T cells or HEK293 cells or alternatively othereukaryotic cells like plant derived cells, fungal or yeast cells. Themethod used to introduce these genes can be methods described in the artsuch as electroporation, lipofectine, lipofectamine or others. After.introduction of these antibody genes in the host cells, cells expressingthe antibody can be identified and selected. These cells represent thetransfectomas which can then be amplified for their expression level andupscaled to produce antibodies. Recombinant antibodies can be isolatedand purified from, these culture supernatants and/or cells.Alternatively, the cloned antibody genes can be expressed in otherexpression systems, including prokaryotic cells, such as microorganisms,e.g. E. coli. Furthermore, the antibodies can be produced in transgenicnon-human animals, such as in milk from sheep and rabbits or in eggsfrom hens, or in transgenic plants; see e.g. Verma, R., et al. (1998) J.Immunol. Meth. 216: 165-181; Pollock, et al. (1999) J. Immunol. Meth.231: 147-157; and Fischer, R., et al. (1999) Biol. Chem. 380: 825-839.

Use of Partial Antibody Sequences to Express Intact Antibodies (i.e.Humanization and Chimerisation).

a) Chimerisation

Murine monoclonal antibodies can be used as therapeutic antibodies inhumans when labeled with toxins or radioactive isotopes. Nonlabeledmurine antibodies are highly immunogenic in man when repetitivelyapplied leading to reduction of the therapeutic effect. The mainimmunogenicity is mediated by the heavy chain constant regions. Theimmunogenicity of murine antibodies in man can be reduced or completelyavoided if respective antibodies are chimerized or humanized. Chimericantibodies are antibodies, the different portions of which are derivedfrom different animal species, such as those having a variable regionderived from a murine antibody and a human immunoglobulin constantregion. Chimerisation of antibodies is achieved by joining of thevariable regions of the marine antibody heavy and light chain with theconstant region of human heavy and light chain (e.g. as described byKraus et al., in Methods in Molecular Biology series, Recombinantantibodies for cancer therapy ISBN-0-89603-918-8). In a preferredembodiment chimeric antibodies are generated by joining humankappa-light chain constant region to murine light chain variable region.In an also preferred embodiment chimeric antibodies can be generated byjoining human lambda-light chain constant region to murine light chainvariable region. The preferred heavy chain constant regions, forgeneration of chimeric antibodies are IgG1, IgG3 and IgG4. Otherpreferred heavy chain constant regions for generation of chimericantibodies are IgG2, IgA, IgD and IgM.

b) Humanization

Antibodies interact with target antigens predominantly through aminoacid residues that are located in the six heavy and light chaincomplementarity determining regions (CDRs). For this reason, the aminoacid sequences within CDRs are more diverse between individualantibodies than sequences outside of CDRs. Because CDR sequences areresponsible for most antibody-antigen interactions, it is possible toexpress recombinant antibodies that mimic the properties of specificnaturally occurring antibodies by constructing expression vectors thatinclude CDR sequences from the specific naturally occurring antibodygrafted onto framework sequences from a different antibody withdifferent properties (see, e.g., Riechmann, L. et al. (1998) Nature 332:323-327; Jones, P. et al. (1986) Nature 321: 522-525; and Queen, C. etal. (1989) Proc. Natl. Acad. Sci. U.S.A. 86: 10029-10033). Suchframework sequences can be obtained from public DNA databases thatinclude germline antibody gene sequences. These germline sequences willdiffer from mature antibody gene sequences because they will not includecompletely assembled variable genes, which are formed by V (D) J joiningduring B cell maturation. Germline gene sequences will also differ fromthe sequences of a high affinity secondary repertoire antibody atindividual evenly across the variable region. For example, somaticmutations are relatively infrequent in the amino terminal portion offramework region 1 and in the carboxy-terminal portion of frameworkregion 4. Furthermore, many somatic mutations do not significantly alterthe binding properties of the antibody. For this reason, it is notnecessary to obtain the entire DNA sequence of a particular antibody inorder to recreate an intact recombinant antibody having bindingproperties similar to those of the original antibody (see WO 99/45962).Partial heavy, and light chain sequences spanning the CDR regions aretypically sufficient for this purpose. The partial sequence is used todetermine which germline variable and joining gene segments contributedto the recombined antibody variable genes. The germline sequence is thenused to fill in missing portions of the variable regions. Heavy andlight chain leader sequences are cleaved during protein maturation anddo not contribute to the properties of the final antibody. To addmissing sequences, cloned cDNA sequences can be combined with syntheticoligonucleotides by ligation or PCR amplification. Alternatively, theentire variable region can be synthesized as a set of short,overlapping, oligonucleotides and combined by PCR amplification tocreate an entirely synthetic variable region clone. This process hascertain advantages such as elimination or inclusion or particularrestriction sites, or optimization of particular codons.

The nucleotide sequences of heavy and light chain transcripts fromhybridomas are used to design an overlapping set of syntheticoligonucleotides to create synthetic V sequences with identical aminoacid coding capacities as the natural sequences. The synthetic heavy andkappa chain sequences can differ from the natural sequences in threeways: strings of repeated nucleotide bases are interrupted to facilitateoligonucleotide synthesis and PCR amplification; optimal translationinitiation sites are incorporated according to Kozak's rules (Kozak,1991, J. Biol. Chem. 266: 19867-19870); and HindIII sites are engineeredupstream of the translation initiation sites.

For both the heavy and light chain variable regions; the optimizedcoding and corresponding non-coding, strand sequences are broken downinto 30-50 nucleotides approximately at the midpoint of thecorresponding non-coding oligonucleotide. Thus, for each chain, theoligonucleotides can be assembled into overlapping double stranded setsthat span segments of 150-400 nucleotides. The pools are then used astemplates to produce PCR amplification products of 150-400 nucleotides.Typically, a single variable region oligonucleotide set will be brokendown into two pools which are separately amplified to generate twooverlapping PCR products. These overlapping products are then combinedby PCR amplification to form the complete variable region. It may alsobe desirable to include an overlapping fragment of the heavy or lightchain constant region, in the PCR amplification to generate fragmentsthat can easily be cloned into the expression vector constructs.

The reconstructed chimerized or humanized heavy and light chain variableregions are then combined with cloned promoter, leader, translationinitiation, constant region, 3′ untranslated, polyadenylation, andtranscription termination sequences to form expression vectorconstructs. The heavy and light chain expression constructs can becombined into a single vector, co-transfected, serially transfected, orseparately transfected into host cells which are then fused to form ahost cell expressing both chains. Plasmids for use in construction ofexpression vectors for human IgGκ are described below. The plasmids wereconstructed so that PCR amplified V heavy and V kappa light chain cDNAsequences could be used to reconstruct complete heavy and light chainminigenes. These plasmids can be used to express completely human, orchimeric IgG1, Kappa or IgG4, Kappa antibodies. Similar plasmids can beconstructed for expression of other heavy chain isotypes, or forexpression of antibodies comprising lambda light chains.

Thus, in another aspect of the invention, the structural features of theanti-CLD18 antibodies of the invention are used to create structurallyrelated humanized anti-CLD18 antibodies that retain at least onefunctional property of the antibodies of the invention, such as bindingto CLD18. More specifically, one or more CDR regions of mouse monoclonalantibodies can be combined recombinantly with known human frameworkregions and CDRs to create additional, recombinantly-engineered,humanized anti-CLD18 antibodies of the invention.

Binding to Antigen Expressing Cells

The ability of the antibody to bind CLD18 can be determined usingstandard binding assays, such as those set forth in the examples (e.g.,ELISA, Western Blot, Immunofluorescence and flow cytometric analysis).

Characterization of Binding of Antibodies

To purify anti-CLD18 antibodies, selected hybridomas can be grown intwo-liter spinner-flasks for monoclonal antibody purification.Alternatively, anti-CLD18 antibodies can be produced in dialysis basedbioreactors. Supernatants can be filtered and, if necessary,concentrated before affinity chromatography with protein G-sepharose orprotein A-sepharose. Eluted IgG can be checked by gel electrophoresisand high performance liquid chromatography to ensure purity. The buffersolution can be exchanged into PBS, and the concentration can bedetermined by OD280 using 1.43 extinction coefficient. The monoclonalantibodies can be aliquoted and stored at −80° C.

To determine if the selected anti-CLD18 monoclonal antibodies bind tounique epitopes, site-directed or multi-site directed mutagenesis can beused.

Isotype Determination

To determine the isotype of purified antibodies, isotype ELISAs withvarious commercial kits (e.g. Zymed, Roche Diagnostics) can beperformed. Wells of microtiter plates can be coated with anti-mouse Ig.After blocking, the plates are reacted with monoclonal antibodies orpurified isotype controls, at ambient temperature for two hours. Thewells can then be reacted with either mouse IgG1, IgG2a, IgG2b or IgG3,IgA or mouse IgM-specific peroxidase-conjugated probes. After washing,the plates can be developed with ABTS substrate (1 mg/ml) and analyzedat OD of 405-650. Alternatively, the IsoStrip Mouse Monoclonal AntibodyIsotyping Kit (Roche, Cat. No. 1493027) may be used as described by themanufacturer.

Flow Cytometric Analysis

In order to demonstrate presence of anti-CLD18 antibodies in sera ofimmunized mice or binding of monoclonal antibodies to living cellsexpressing CLD18, flow cytometry can be used. Cell lines expressingnaturally or after. transfection CLD18 and negative controls lackingCLD18 expression (grown under standard growth conditions) can be mixedwith various concentrations of monoclonal antibodies in hybridomasupernatants or in PBS containing 1% FBS, and can be incubated at 4° C.for 30 rain. After washing, the APC- or Alexa647-labeled anti IgGantibody can bind to CLD18-bound monoclonal antibody under the sameconditions as the primary antibody staining. The samples can be analyzedby flow cytometry with a FACS instrument using light and side scatterproperties to gate on single, living cells. In order to distinguishCLD18-specific monoclonal antibodies from non-specific binders in asingle measurement, the method of co-transfection can be employed. Cellstransiently transfected with plasmids encoding CLD18 and a fluorescentmarker can be stained as described above. Transfected cells can bedetected in a different fluorescence channel than antibody-stainedcells. As the majority of transfected cells express both transgenes,CLD18-specific monoclonal antibodies bind preferentially to fluorescencemarker expressing cells, whereas non-specific antibodies bind in acomparable ratio to non-transfected cells. An alternative assay usingfluorescence microscopy may be used in addition to or instead of theflow cytometry assay. Cells can be stained exactly as described aboveand examined by fluorescence, microscopy.

Tight junction proteins tend to be internalized, if cell contact toneighbouring cells of particularly adherent cells is lost by e.g.detachment of cells. Cell surface expression of CLD18 can be optimizedby a) adjusting culture conditions, e.g. culturing in higher celldensity in a standardized manner, using mild detachment (e.g. 2 mMEDTA/PBS or accutase), processing at room temperature, and addinginhibitors of endocytosis (e.g. sodium azide) or activators of CLD18transcription or translation, and by b) selecting and cloning of cellsmaintaining CLD18 in high levels at the cell surface, e.g. by selectionwith antibiotics in terms of transfected cells, by immunomagnetic orFACS cell sorting, and by limited dilution cloning.

Immunofluorescence Microscopy

In order to demonstrate presence of anti-CLD18 antibodies in sera ofimmunized mice or binding of monoclonal antibodies to living cellsexpressing CLD18, immunofluorescence microscopy analysis can be used.For example, cell lines expressing either spontaneously or aftertransfection CLD18 and negative controls lacking CLD18 expression aregrown in chamber slides under standard growth conditions in DMEM/F′12medium, supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine,100 IU/ml penicillin and 100 μg/ml streptomycin. Cells can then be fixedwith methanol or paraformaldehyde or left untreated. Cells can then bereacted with monoclonal antibodies against CLD18 for 30 min. at 25° C.After washing, cells can be reacted with an Alexa555-labelled anti-mouseIgG secondary antibody (Molecular Probes) under the same conditions.Cells can then be examined by fluorescence microscopy.

Total CLD18 levels in cells can be observed when cells are methanolfixed or paraformaldehyde fixed and permeabilized with Triton X-100. Inliving cells and non-permeabilized, paraformaldehyde fixed cells surfacelocalization of CLD18 can be examined. Additionally targeting of CLD18to tight junctions can be analyzed by co-staining with tight junctionmarkers such as ZO-1. Furthermore, effects of antibody binding and CLD18localization within the cell membrane can be examined.

Western Blot

Anti-CLD18 IgG can be further tested for reactivity with CLD18 antigenby Western Blotting. Briefly, cell extracts from cells expressing CLD18and appropriate negative controls can be prepared and subjected tosodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Afterelectrophoresis, the separated antigens will be transferred tonitrocellulose membranes, blocked, and probed with the monoclonalantibodies to be tested. IgG binding can be detected using anti-mouseIgG peroxidase and developed with ECL substrate.

Immunohistochemistry

Anti-CLD18 mouse IgGs can be further tested for reactivity with CLD18antigen by Immunohistochemistry in a manner well known to the skilledperson, e.g. using paraformaldehyde or acetone fixed cryosections orparaffin embedded tissue sections fixed with paraformaldehyde fromnon-cancer tissue or cancer tissue samples obtained from patients duringroutine surgical procedures or from mice carrying xenografted tumorsinoculated with cell lines expressing spontaneously (e.g. DAN-G, SNU-16,or KATO-III) or after transfection (e.g. HEK293) CLD18. Forimmunostaining antibodies reactive to CLD18 can be incubated followed byhorseradish-peroxidase conjugated goat anti-mouse or goat anti-rabbitantibodies (DAKO) according to the vendors instructions.

Phagocytic and Cell Killing Activities of Antibodies In Vitro

In addition to binding specifically to CLD18, anti-CLD18 antibodies canbe tested for their ability to mediate phagocytosis and killing of cellsexpressing CLD18. The testing of monoclonal antibody activity in vitrowill provide an initial screening prior to testing in vivo models.

Antibody Dependent Cell-Mediated Cytotoxicity (ADCC):

Briefly, polymorphonuclear cells (PMNs), NK cells, monocytes,mononuclear cells or other effector cells, from healthy donors can bepurified by Ficoll Hypaque density centrifugation, followed by lysis ofcontaminating erythrocytes. Washed effector cells can be suspended inRPMI supplemented with 10% heat-inactivated fetal calf serum or,alternatively with 5% heat-inactivated human serum and mixed with ⁵¹Crlabeled target cells expressing CLD18, at various ratios of effectorcells to target cells. Alternatively, the target cells may be labeledwith a fluorescence enhancing ligand (BATDA). A highly fluorescentchelate of Europium with the enhancing ligand which is released fromdead cells can be measured by a fluorometer. Another alternativetechnique may utilize the transfection of target cells with luciferase.Added lucifer yellow may then be oxidated by viable cells only. Purifiedanti-CLD18 IgGs can then be added at various concentrations. Irrelevanthuman IgG can be used as negative control. Assays can be carried out for4 to 20 hours at 37° C. depending on the effector cell type used.Samples can be assayed for cytolysis by measuring ⁵¹Cr release or thepresence of the EuTDA chelate in the culture supernatant. Alternatively,luminescence resulting from the oxidation of lucifer yellow can be ameasure of viable cells.

Anti-CLD18 monoclonal antibodies can also be tested in variouscombinations to determine whether cytolysis is enhanced with multiplemonoclonal antibodies.

Complement Dependent Cytotoxicity (CDC):

Monoclonal anti-CLD18 antibodies can be tested for their ability tomediate CDC using a variety of known techniques. For example, serum forcomplement can be obtained from blood in a manner known to the skilledperson. To determine the CDC activity of mAbs, different methods can beused. ⁵¹Cr release can for example be measured or elevated membranepermeability can be assessed using a propidium iodide (PI) exclusionassay. Briefly, target cells can be washed and 5×10⁵/ml can be incubatedwith various concentrations of mAb for 10-30 min. at room temperature orat 37° C. Serum or plasma can then be added to a final concentration of20% (v/v) and the cells incubated at 37° C. for 20-30 min. All cellsfrom each sample can be added to the PI solution in a FACS tube. Themixture can then be analyzed immediately by flow cytometry analysisusing FACSArray. In an alternative assay, induction of CDC can bedetermined on adherent cells. In one embodiment of this assay, cells areseeded 24 h before the assay with a density of 3×10⁴/well intissue-culture flat-bottom microliter plates. The next day growth mediumis removed and the cells are incubated in triplicates with antibodies.Control cells are incubated with growth medium or growth mediumcontaining 0.2% saponin for the determination of background lysis andmaximal lysis, respectively. After incubation for 20 min. at roomtemperature supernatant is removed and 20% (v/v) human plasma or serumin DMEM (prewarmed to 37° C.) is added to the cells and incubated foranother 20 min. at 37° C. All cells from each sample are added topropidium iodide solution (10 μg/ml). Then, supernatants are replaced byPBS containing 2.5 ethidium bromide and fluorescence emission uponexcitation at 520 nm is measured at 600 nm using a Tecan Safire. Thepercentage specific lysis is calculated as follows: % specificlysis=(fluorescence sample−fluorescence background)/(fluorescencemaximal lysis−fluorescence background)×100.

Inhibition of Cell Proliferation by Monoclonal Antibodies:

To test for the ability to initiate apoptosis, monoclonal anti-CLD18antibodies can, for example, be incubated with CLD18 positive tumorcells, e.g., SNU-16, DAN-G, KATO-III or CLD18 transfected tumor cells at37° C. for about 20 hours. The cells can be harvested, washed inAnnexin-V binding, buffer (BD biosciences), and incubated with Annexin Vconjugated with FITC or APC (BD biosciences) for 15 min. in the dark.All cells from each sample can be added to PI solution (10 μg/ml in PBS)in a FACS tube and assessed immediately by flow cytometry (as above).Alternatively, a general inhibition of cell-proliferation by monoclonalantibodies can be detected with commercially available kits. The DELFIACell Proliferation Kit (Perkin-Elmer, Cat. No. AD0200) is a non-isotopicimmunoassay based on the measurement of 5-bromo-2′-deoxyuridine (BrdU)incorporation during DNA synthesis of proliferating cells inmicroplates. Incorporated BrdU is detected using europium labelledmonoclonal antibody. To allow antibody detection, cells are fixed andDNA denatured using Fix solution. Unbound antibody is washed away andDELFIA inducer is added to dissociate europium ions from the labeledantibody into solution, where they form highly fluorescent chelates withcomponents of the DELFIA Inducer. The fluorescence measured—utilizingtime resolved fluorometry in the detection—is proportional to the DNAsynthesis in the cell of each well.

Preclinical Studies

Monoclonal antibodies which bind to CLD18 also can be tested in an invivo model (e.g. in immune deficient mice carrying xenografted tumorsinoculated with cell lines expressing CLD18, e.g. DAN-G, SNU-16, orKATO-III, or after transfection, e.g. HEK293) to determine theirefficacy in controlling growth of CLD18-expressing tumor cells.

In vivo studies after xenografting CLD18 expressing tumor cells intoimmunocompromised mice or other animals can be performed usingantibodies of the invention. Antibodies can be administered to tumorfree mice followed by injection of tumor cells to measure the effects ofthe antibodies to prevent formation of tumors or tumor-related symptoms.Antibodies can be administered to tumor-bearing mice to determine thetherapeutic efficacy of respective antibodies to reduce tumor growth,metastasis or tumor related symptoms. Antibody application can becombined with application of other substances as cystostatic drugs,growth factor inhibitors, cell cycle blockers, angiogenesis inhibitorsor other antibodies to determine synergistic efficacy and potentialtoxicity of combinations. To analyze toxic side effects mediated byantibodies of the invention animals can be inoculated with antibodies orcontrol reagents and thoroughly investigated for symptoms possiblyrelated to CLD18-antibody therapy. Possible side effects of in vivoapplication of CLD18 antibodies particularly include toxicity at CLD18expressing tissues including stomach and lung. Antibodies recognizingCLD18 in human and in other species, e.g. mice, are particularly usefulto predict potential side effects mediated by application of monoclonalCLD18-antibodies in humans.

Epitope Mapping

Mapping of epitopes recognized by antibodies of invention can beperformed as described in detail in “Epitope Mapping Protocols” (Methodsin Molecular Biology) by Glenn E. Morris ISBN-089603-375-9 and in“Epitope Mapping: A Practical Approach” Practical Approach Series, 248by Olwyn M. R. Westwood, Frank C. Hay.

I. Bispecific/Multispecific Molecules which Bind to CLD18

In yet another embodiment of the invention, antibodies to CLD18 can bederivatized or linked to another functional molecule, e.g., anotherpeptide or protein (e.g., an Fab′ fragment) to generate a bispecific ormultispecific molecule which binds to multiple binding sites or targetepitopes. For example, an antibody of the invention can be functionallylinked (e.g. by chemical coupling, genetic fusion, noncovalentassociation or otherwise) to one or more other binding molecules, suchas another antibody, peptide or binding mimetic.

Accordingly, the present invention includes bispecific and multispecificmolecules comprising at least one first binding specificity for CLD18and a second binding specificity for a second target epitope. In aparticular embodiment of the invention, the second target epitope is anFc receptor, e.g. human Fc-gammaRI (CD64) or a human Fc-alpha receptor(CD89), or a T cell receptor, e.g. CD3. Therefore, the inventionincludes bispecific and multispecific molecules capable of binding bothto Fc-gammaR, Fc-alphaR or Fc-epsilonR expressing effector cells (e.g.monocytes, macrophages or polymorphonuclear cells (PMNs)), and to targetcells expressing CLD18. These bispecific and multispecific molecules maytarget CLD18 expressing cells to effector cell and may trigger Fcreceptor-mediated effector cell activities, such as phagocytosis ofCLD18 expressing cells, antibody dependent cellular cytotoxicity (ADCC),cytokine release, or generation of superoxide anion.

Bispecific and multispecific molecules of the invention can furtherinclude a third binding specificity, in addition to an anti-Fc bindingspecificity and an anti-CLD18 binding specificity. In one embodiment,the third binding specificity is an anti-enhancement factor (EF)portion, e.g. a molecule which binds to a surface protein involved incytotoxic activity and thereby increases the immune response against thetarget cell. The “anti-enhancement factor portion” can be an antibody,functional antibody fragment or a ligand that binds to a given molecule,e.g., an antigen or a receptor, and thereby results in an enhancement ofthe effect of the binding determinants for the Fc receptor or targetcell antigen. The “anti-enhancement factor portion” can bind an Fcreceptor or a target cell antigen. Alternatively, the anti-enhancementfactor portion can bind to an entity that is different from the entityto which the first and second binding specificities bind. For example,the anti-enhancement factor portion can bind a cytotoxic T cell (e.g.,via CD2, CD3, CD8, CD28, CD4, CD40, ICAM-1 or other immune cell thatresults in an increased immune response against the target cell).

In one embodiment, the bispecific and multispecific molecules of theinvention comprise as a binding specificity at least one antibody,including, e.g., an Fab, Fab′, F(ab′)₂, Fv, or a single chain Fv. Theantibody may also be a light chain or heavy chain dimer, or any minimalfragment thereof such as a Fv or a single chain construct as describedin Ladner et al., U.S. Pat. No. 4,946,778. The antibody may also be abinding-domain immunoglobulin fusion protein as disclosed inUS2003/0118592 and US 2003/0133939.

In one embodiment bispecific and multispecific molecules of theinvention comprise a binding specificity for an Fc-gammaR or anFc-alphaR present on the surface of an effector cell, and a secondbinding specificity for a target cell antigen, e.g., CLD18.

In one embodiment, the binding specificity for an Fc receptor isprovided by a monoclonal antibody, the binding of which is not blockedby human immunoglobulin G (IgG). As used herein, the team “IgG receptor”refers to any of the eight gamma-chain genes located on chromosome 1.These genes encode a total of twelve transmembrane or soluble receptorisoforms which are grouped into three Fc-gamma receptor classes:Fc-gammaRI (CD64), Fc-gammaRII (CD32), and Fc-gammaRIII (CD16). In onepreferred embodiment, the Fc-gamma receptor is a human high affinityFc-gammaRI.

The production and characterization of these preferred monoclonalantibodies are described by Fanger et al. in WO 88/00052 and in U.S.Pat. No. 4,954,617. These antibodies bind to an epitope of Fc-gammaRI,Fc-gammaRII or Fc-gammayRIII at a site which is distinct from the Fcγbinding site of the receptor and, thus, their binding is not blockedsubstantially by physiological levels of IgG. Specific anti-Fc gammaRIantibodies useful in this invention are mAb 22, mAb 32, mAb 44, mAb 62and mAb 197. In other embodiments, the anti-Fey receptor antibody is ahumanized form of monoclonal antibody 22 (H22). The production andcharacterization of the H22 antibody is described in Graziano, R. F. etal. (1995) J. Immunol. 155 (10): 4996-5002 and WO 94/10332. The H22antibody producing cell line was deposited at the American Type CultureCollection on Nov. 4, 1992 under the designation HA022CL1 and has theaccession No. CRL 11177.

In still other preferred embodiments, the binding specificity for an Fcreceptor is provided by an antibody that binds to a human IgA receptor,e.g., an Fc-alpha receptor (Fc-alphaRI (CD89)), the binding of which ispreferably not blocked by human immunoglobulin A (IgA). The team “IgAreceptor” is intended to include the gene product of one alpha-gene(Fc-alphaRI) located on chromosome 19. This gene is known to encodeseveral alternatively spliced transmembrane isoforms of 55 to 110 kDa.Fc-alphaRI (CD89) is constitutively expressed on monocytes/macrophoges,eosinophilic and neutrophilic granulocytes, but not on non-effector cellpopulations. Fc-alphaRI has medium affinity for both IgA1 and IgA2,which is increased upon exposure to cytokines such as G-CSF or GM-CSF(Morton, H. C. et al. (1996) Critical Reviews in Immunology 16:423-440). Four Fc-alphaRI-specific monoclonal antibodies, identified asA3, A59, A62 and A77, which bind Fc-alphaRI outside the IgA ligandbinding domain, have been described (Monteiro, R. C. et al. (1992) J.Immunol. 148: 1764).

Fc-alphaRI and Fc-gammaRI are preferred trigger receptors for use in theinvention because they (1) are expressed primarily on immune effectorcells, e.g., monocytes, PMNs, macrophages and dendritic cells; (2) areexpressed at high levels (e.g., 5,000-100,000 per cell); (3) aremediators of cytotoxic activities (e.g., ADCC, phagocytosis); (4)mediate enhanced antigen presentation of antigens, includingself-antigens, targeted to them.

In another embodiment the bispecific molecule is comprised of twomonoclonal antibodies according to the invention which havecomplementary functional activities, such as one antibody predominatelyworking by inducing CDC and the other antibody predominately working byinducing apoptosis.

An “effector cell specific antibody” as used herein refers to anantibody or functional antibody fragment that binds the Fc receptor ofeffector cells. Preferred antibodies for use in the subject inventionbind the Fc receptor of effector cells at a site which is not bound byendogenous immunoglobulin.

As used herein, the term “effector cell” refers to an immune cell whichis involved in the effector phase of an immune response, as opposed tothe cognitive and activation phases of an immune response. Exemplaryimmune cells include cells of myeloid or lymphoid origin, e.g,lymphocytes (e.g., B cells and T cells including cytolytic T cells(CTLs), killer cells, natural killer cells, macrophages, monocytes,eosinophils, neutrophils, polymorphonuclear cells, granulocytes, mastcells, and basophils. Some effector cells express specific Fc receptorsand carry out specific immune functions. In preferred embodiments, aneffector cell is capable of inducing antibody-dependent cellularcytotoxicity (ADCC), e.g., a neutrophil capable of inducing ADCC. Forexample, monocytes, macrophages, which express FcR are involved inspecific killing of target cells and presenting antigens to othercomponents of the immune system, or binding to cells that presentantigens. In other embodiments, an effector cell can phagocytose atarget antigen, target cell, or microorganism. The expression of aparticular FcR on an effector cell can be regulated by humoral factorssuch as cytokines. For example, expression of Fc-gammaRI has been foundto be up-regulated by interferon gamma (IFN-γ). This enhanced expressionincreases the cytotoxic activity of Fc-gammaR1-bearing cells againsttargets. An effector cell can phagocytose or lyse a target antigen or atarget cell.

“Target cell” shall mean any undesirable cell in a subject (e.g., ahuman or animal) that can be targeted by an antibody of the invention.In preferred embodiments, the target cell is a cell expressing oroverexpressing CLD18. Cells expressing CLD18 typically include tumorcells.

Bispecific and multispecific molecules of the present invention can bemade using chemical techniques (see e.g., D. M. Kranz et al. (1981)Proc. Natl. Acad. Sci. USA 78:5807), “polydoma” techniques (See U.S.Pat. No. 4,474,893, to Reading), or recombinant DNA techniques.

In particular, bispecific and multispecific molecules of the presentinvention can be prepared by conjugating the constituent bindingspecificities, e.g., the anti-FcR and anti-CLD18 binding specificities,using methods known in the art. For example, each binding specificity ofthe bispecific and multispecific molecule can be generated separatelyand then conjugated to one another. When the binding specificities areproteins or peptides, a variety of coupling or cross-linking agents canbe used for covalent conjugation. Examples of cross-linking agentsinclude protein A, carbodiimide, N-succinimidyl-5-acetyl-thioacetate(SATA), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB),o-phenylenedimaleimide (oPDM),N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), andsulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate(sulfo-SMCC) (see e.g., Karpovsky et al. (1984) J. Exp. Med. 160: 1686;Liu, M A et al. (1985) Proc. Natl. Acad. Sci. USA 82: 8648). Othermethods include those described by Paulus (Behring Ins. Mitt. (1985) No.78, 118-132); Brennan et al. (Science (1985) 229: 81-83), and Glennie etal. (J. Immunol. (1987) 139: 2367-2375). Preferred conjugating agentsare SATA and sulfo-SMCC, both available from Pierce Chemical Co.(Rockford, Ill.).

When the binding specificities are antibodies, they can be conjugatedvia sulfhydryl bonding of the C-terminus hinge regions of the two heavychains. In a particularly preferred embodiment, the hinge region ismodified to contain an odd number of sulfhydryl residues, preferablyone, prior to conjugation.

Alternatively, both binding specificities can be encoded in the samevector and expressed and assembled in the same host cell. This method isparticularly useful where the bispecific and multispecific molecule is amAb×mAb, mAb×Fab, Fab×F(ab′₂) or ligand×Fab fusion protein. A bispecificand multispecific molecule of the invention, e.g., a bispecificmolecule, can be a single chain molecule, such as a single chainbispecific antibody, a single chain bispecific molecule comprising onesingle chain antibody and a binding determinant, or a single chainbispecific molecule comprising two binding determinants. Bispecific andmultispecific molecules can also be single chain molecules or maycomprise at least two single chain molecules. Methods for preparing bi-and multispecific molecules are described for example in U.S. Pat. No.5,260,203; U.S. Pat. No. 5,455,030; U.S. Pat. No. 4,881,175; U.S. Pat.No. 5,132,405; U.S. Pat. No. 5,091,513; U.S. Pat. No. 5,476,786; U.S.Pat. No. 5,013,653; U.S. Pat. No. 5,258,498; and U.S. Pat. No.5,482,858.

Binding of the bispecific and multispecific molecules to their specifictargets can be confirmed by enzyme-linked immunosorbent assay (ELISA), aradioimmunoassay (RIA), FACS analysis, a bioassay (e.g., growthinhibition), or a Western Blot Assay. Each of these assays generallydetects the presence of protein-antibody complexes of particularinterest by employing a labeled reagent (e.g., an antibody) specific forthe complex of interest. For example, the FcR-antibody complexes can bedetected using e.g., an enzyme-linked antibody or antibody fragmentwhich recognizes and specifically binds to the antibody-FcR complexes.Alternatively, the complexes can be detected using any of a variety ofother immunoassays. For example, the antibody can be radioactivelylabeled and used in a radioimmunoassay (RIA) (see, for example,Weintraub, B., Principles of Radioimxnunoassays, Seventh Training Courseon Radioligand Assay Techniques, The Endocrine Society, March, 1986).The radioactive isotope can be detected by such means as the use of aγ-counter or a scintillation counter or by autoradiography.

II. Immunoconjugates

In another aspect, the present invention features an anti-CLD18 antibodyconjugated to a therapeutic moiety or agent, such as a cytotoxia, a drug(e.g., an immunosuppressant) or a radioisotope. Such conjugates arereferred to herein as “Immunoconjugates”. Immunoconjugates which includeone or more cytotoxins are referred to as “immunotoxins”. A cytotoxin orcytotoxic agent includes any agent that is detrimental to and, inparticular, kills cells. Examples include taxol, cytochalasin B,gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin and analogs orhomologs thereof.

Suitable therapeutic agents for forming immunoconjugates of theinvention include, but are not limited to, antimetabolites (e.g.,methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabin,5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine,thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cis-dichlorodiamine platinum (II) (DDP) cisplatin),anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC), and anti-mitotic agents(e.g., vincristine and vinblastine). In a preferred embodiment, thetherapeutic agent is a cytotoxic agent or a radiotoxic agent. In anotherembodiment, the therapeutic agent is an immunosuppressant. In yetanother embodiment, the therapeutic agent is GM-CSF. In a preferredembodiment, the therapeutic agent is doxorubicin, cisplatin, bleomycin,sulfate, carmustine, chlorambucil, cyclophosphamide or ricin A.

Antibodies of the present invention also can be conjugated to aradioisotope, e.g., iodine-131, yttrium-90 or indium-111, to generatecytotoxic radiopharmaceuticals for treating a CLD18-related disorder,such as a cancer. The antibody conjugates of the invention can be usedto modify a given biological response, and the drug moiety is not to beconstrued as limited to classical chemical therapeutic agents. Forexample, the drug moiety may be a protein or polypeptide possessing adesired biological activity. Such proteins may include, for example, anenzymatically active toxin, or active fragment thereof, such as abrin,ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such astumor necrosis factor or interferon-γ; or, biological response modifierssuch as, for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2(“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colonystimulating factor (“GM-CSF”), granulocyte colony stimulating factor(“G-CSF”), or other growth factors.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Amon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Helistrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev., 62: 119-58 (1982).

In a further embodiment, the antibodies according to the invention areattached to a linker-chelator, e.g., tiuxetan, which allows for theantibody to be conjugated to a radioisotope.

III. Pharmaceutical Compositions

In another aspect, the present invention provides a composition, e.g., apharmaceutical composition, containing one or a combination ofantibodies of the present invention. The pharmaceutical compositions maybe formulated with pharmaceutically acceptable carriers or diluents aswell as any other known adjuvants and excipients in accordance withconventional techniques such as those disclosed in Remington: TheScience and Practice of Pharmacy, 19th Edition, Gennaro, Ed., MackPublishing Co., Easton, Pa., 1995. In one embodiment, the compositionsinclude a combination of multiple (e.g., two or more) isolatedantibodies of the invention which act by different mechanisms, e.g., oneantibody which predominately acts by inducing CDC in combination withanother antibody which predominately acts by inducing apoptosis.

Pharmaceutical compositions of the invention also can be administered incombination therapy, i.e., combined with other agents. For example, thecombination therapy can include a composition of the present inventionwith at least one anti-inflammatory agent or at least oneimmunosuppressive agent. In one embodiment such therapeutic agentsinclude one or more anti-inflammatory agents, such as a steroidal drugor a NSAID (nonsteroidal anti-inflammatory drug). Preferred agentsinclude, for example, aspirin and other salicylates, Cox-2 inhibitors,such as rofecoxib (Vioxx) and celecoxib (Celebrex), NSAIDs such asibuprofen (Motrin, Advil), fenoprofen (Nalfon), naproxen (Naprosyn),sulindac (Clinoril), diclofenac (Voltaren), piroxicam (Feldene),ketoprofen (Orudis), difhinisal (Dolobid), nabumetone (Relafen),etodolac (Lodine), oxaprozin (Daypro), and indomethacin (Indocin).

In another embodiment, such therapeutic agents include agents leading tothe depletion or functional inactivation of regulatory T cells like lowdose cyclophosphamid, anti-CTLA4 antibodies, anti-IL2 oranti-IL2-receptor antibodies.

In yet another embodiment, such therapeutic agents, include one or morechemotherapeutics, such as Taxol derivatives, taxotere, gemcitabin,5-Fluoruracil, doxorubicin (Adriamycin), cisplatin (Platinol),cyclophosphamide (Cytoxan, Procytox, Neosar). In another embodiment,antibodies of the present invention may be administered in combinationwith chemotherapeutic agents, which preferably show therapeutic efficacyin patients suffering from stomach, esophageal, pancreatic and lungcancer.

In yet another embodiment, the antibodies of the invention may beadministered in conjunction with radiotherapy and/or autologousperipheral stem cell or bone marrow transplantation.

In still another embodiment, the antibodies of the invention may beadministered in combination with one or more antibodies selected fromanti-CD25 antibodies, anti-EPCAM antibodies, anti-EGFR, anti-Her2/neu,and anti-CD40 antibodies.

In yet a further embodiment, the antibodies of the invention may beadministered in combination with an anti-C3b(i) antibody in order toenhance complement activation.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Preferably, the carrier is suitable forintravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g., by injection or infusion). Depending onthe route of administration, the active compound, i.e., antibody,bispecific and multispecific molecule, may be coated in a material toprotect the compound from the action of acids and other naturalconditions that may inactivate the compound.

A “pharmaceutically acceptable salt” refers to a salt that retains thedesired biological activity of the parent compound and does not impartany undesired toxicological effects (see e.g., Berge, S. M., et al.(1977) J. Pharm. Sci. 66: 1-19).

Examples of such salts include acid addition salts and base additionsalts. Acid addition salts include those derived from nontoxic inorganicacids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic,hydroiodic, phosphorous and the like, as well as from nontoxic organicacids such as aliphatic mono- and dicarboxylic acids, phenyl-substitutedalkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic andaromatic sulfonic acids and the like. Base addition salts include thosederived from alkaline earth metals, such as sodium, potassium,magnesium, calcium and the like, as well as from nontoxic organicamines, such as N,N′-dibenzylethylenediamine, N-methylglucamine,chloroprocaine, choline, diethanolamine, ethylenediamine, procaine andthe like.

A composition of the present invention can be administered by a varietyof methods known in the art. As will be appreciated by the skilledartisan, the route and/or mode of administration will vary dependingupon the desired results. The active compounds can be prepared withcarriers that will protect the compound against rapid release, such as acontrolled release formulation, including implants, transdermal patches,and microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for the preparation of such formulations are generally known tothose skilled in the art. See, e.g., Sustained and Controlled ReleaseDrug Delivery. Systems, J. R. Robinson, ed., Marcel Dekker, Inc., NewYork, 1978.

To administer a compound of the invention by certain routes ofadministration, it may be necessary to coat the compound with, orco-administer the compound with, a material to prevent its inactivation.For example, the compound may be administered to a subject in anappropriate carrier, for example, liposomes, or a diluent.Pharmaceutically acceptable diluents include saline and aqueous buffersolutions. Liposomes include water-in-oil-in-water CGF emulsions as wellas conventional liposomes (Strejan et al. (1984) J. Neuroimmunol. 7:27).

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersions. The use of such media andagents for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the pharmaceutical compositions ofthe invention is contemplated. Supplementary active compounds can alsobe incorporated into the compositions.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The carrier can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, or sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate salts and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed bysterilization microfiltration.

Generally, dispersions are prepared by incorporating the active compoundinto a sterile vehicle that contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-drying(lyophilization) that yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the active compound andthe particular therapeutic effect to be achieved, and (b) thelimitations inherent in the art of compounding such an active compoundfor the treatment of sensitivity in individuals.

Examples of pharmaceutically-acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

For the therapeutic compositions, formulations of the present inventioninclude those suitable for oral, nasal, topical (including buccal andsublingual), rectal, vaginal and/or parenteral administration. Theformulations may conveniently be presented in unit dosage form and maybe prepared by any methods known in the art of pharmacy. The amount ofactive ingredient which can be combined with a carrier material toproduce a single dosage form will vary depending upon the subject beingtreated, and the particular mode of administration. The amount of activeingredient which can be combined with a carrier material to produce asingle dosage form will generally be that amount of the compositionwhich produces a therapeutic effect.

Generally, out of one hundred percent, this amount will range from about0.01 percent to about ninety-nine percent of active ingredient,preferably from about 0.1 percent to about 70 percent, most preferablyfrom about 1 percent to about 30 percent.

Formulations of the present invention which are suitable for vaginaladministration also include pessaries, tampons, creams, gels, pastes,foams or spray formulations containing such carriers as are known in theart to be appropriate. Dosage forms for the topical or transdermaladministration of compositions of this invention include powders,sprays, ointments, pastes, creams, lotions, gels, solutions, patches andinhalants. The active compound may be mixed under sterile conditionswith a pharmaceutically acceptable carrier, and with any preservatives,buffers, or propellants which may be required.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, epidural and intrasternal injection andinfusion.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe presence of microorganisms may be ensured both by sterilizationprocedures, and by the inclusion of various antibacterial and antifungalagents, for example, paraben, chlorobutanol, phenol sorbic acid, and thelike. It may also be desirable to include isotonic agents, such assugars, sodium chloride, and the like into the compositions. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents which delay absorption suchas aluminum monostearate and gelatin.

In one embodiment the monoclonal antibodies of the invention areadministered in crykalline form by subcutaneous injection, cf. Yang etal. (2003) PNAS, 100 (12): 6934-6939. When the compounds of the presentinvention are administered as pharmaceuticals, to humans and animals,they can be given alone or as a pharmaceutical composition containing,for example, 0.01 to 99.5% (more preferably, 0.1 to 90%) of activeingredient in combination with pharmaceutically acceptable carrier.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically acceptable dosage forms by conventional methodsknown to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level will depend upon a variety of pharmacokinetic factorsincluding the activity of the particular compositions of the presentinvention employed, the route of administration, the time ofadministration, the rate of excretion of the particular compound beingemployed, the duration of the treatment, other drugs, compounds and/ormaterials used in combination with the particular compositions employed,the age, sex, weight, condition, general health and prior medicalhistory of the patient being treated, and like factors well known in themedical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the compounds of the invention employed in thepharmaceutical composition at levels lower than that required in orderto achieve the desired therapeutic effect and gradually increase thedosage until the desired effect is achieved. In general, a suitabledaily dose of a composition of the invention will be that amount of thecompound which is the lowest dose effective to produce a therapeuticeffect. Such an effective dose will generally depend upon the factorsdescribed above. It is preferred that administration be intravenous,intramuscular, intraperitoneal, or subcutaneous, preferably administeredproximal to the site of the target. If desired, the effective daily doseof a therapeutic composition may be administered as two, three, four,five, six or more sub-doses administered separately at appropriateintervals throughout the day, optionally, in unit dosage forms. While itis possible for a compound of the present invention to be administeredalone, it is preferable to administer the compound as a pharmaceuticalformulation (composition).

In one embodiment, the antibodies of the invention may be administeredby infusion, preferably slow continuous infusion over a long period,such as more than 24 hours, in order to reduce toxic side effects. Theadministration may also be performed by continuous infusion over aperiod of from 2 to 24 hours, such as of from 2 to 12 hours. Suchregimen may be repeated one or more times as necessary, for example,after 6 months or 12 months. The dosage can be determined or adjusted bymeasuring the amount of circulating monoclonal anti-CLD18 antibodiesupon administration in a biological sample by using anti-idiotypicantibodies which target the anti-CLD18 antibodies.

In yet another embodiment, the antibodies are administered bymaintenance therapy, such as, e.g., once a week for a period of 6 monthsor more.

In still another embodiment, the antibodies according to the inventionmay be administered by a regimen including one infusion of an antibodyagainst CLD18 followed by an infusion of an antibody against CLD18conjugated to a radioisotope. The regimen may be repeated, e.g., 7 to 9days later.

Therapeutic compositions can be administered with medical devices knownin the art. For example, in a preferred embodiment, a therapeuticcomposition of the invention can be administered with a needlelesshypodermic injection device, such as the devices disclosed in U.S. Pat.No. 5,399,163; U.S. Pat. No. 5,383,851; U.S. Pat. No. 5,312,335; U.S.Pat. No. 5,064,413; U.S. Pat. No. 4,941,880; U.S. Pat. No. 4,790,824; orU.S. Pat. No. 4,596,556. Examples of well-known implants and modulesuseful in the present invention include those described in: U.S. Pat.No. 4,487,603, which discloses an implantable micro-infusion pump fordispensing medication at a controlled rate; U.S. Pat. No. 4,486,194,which discloses a therapeutic device for administering mendicantsthrough the skin; U.S. Pat. No. 4,447,233, which discloses a medicationinfusion pump for delivering medication at a precise infusion rate; U.S.Pat. No. 4,447,224, which discloses a variable flow implantable infusionapparatus for continuous drug delivery; U.S. Pat. No. 4,439,196, whichdiscloses an osmotic drug delivery system having multi-chambercompartments; and U.S. Pat. No. 4,475,196, which discloses an osmoticdrug delivery system.

Many other such implants, delivery systems, and modules are known tothose skilled in the art. In certain embodiments, the antibodies of theinvention can be formulated to ensure proper distribution in vivo. Forexample, the blood-brain barrier (BBB) excludes many highly hydrophiliccompounds. To ensure that the therapeutic compounds of the inventioncross the BBB (if desired), they can be formulated, for example, inliposomes. For methods of manufacturing liposomes, see, e.g., U.S. Pat.No. 4,522,811; U.S. Pat. No. 5,374,548; and U.S. Pat. No. 5,399,331. Theliposomes may comprise one or more moieties which are selectivelytransported into specific cells or organs, and thus enhance targeteddrug delivery (see, e.g., V.V. Ranade (1989) J. Clin. Pharmacol. 29:685). Exemplary targeting moieties include folate or biotin (see, e.g.,U.S. Pat. No. 5,416,016 to Low et al.); mannosides (Umezawa et al.,(1988) Biochem. Biophys. Res. Commun. 153: 1038); antibodies (P. G.Bloeman et al. (1995) FEBS Lett. 357: 140; M. Owais et al. (1995)Antimicrob. Agents Chemother. 39: 180); and surfactant protein Areceptor (Briscoe et al. (1995) Am. J. Physiol. 1233: 134).

In one embodiment of the invention, the therapeutic compounds of theinvention are formulated in liposomes. In a more preferred embodiment,the liposomes include a targeting moiety. In a most preferredembodiment, the therapeutic compounds in the liposomes are delivered bybolus injection to a site proximal to the desired area, e.g., the siteof a tumor. The composition must be fluid to the extent thatsyringability exists. It must be stable under the conditions ofmanufacture and storage and must be preserved against the contaminatingaction of microorganisms such as bacteria and fungi.

In a further embodiment, antibodies of the invention can be formulatedto prevent or reduce their transport across the placenta. This can bedone by methods known in the art, e.g., by PEGylation of the antibodiesor by use of F(ab)₂′ fragments.

Further references can be made to “Cunningham-Rundles C, Zhuo Z,Griffith B, Keenan J. (1992) Biological activities ofpolyethylene-glycol immunoglobulin conjugates. Resistance to enzymaticdegradation. J. Immunol. Methods, 152: 177-190; and to “Landor M. (1995)Maternal-fetal transfer of immunoglobulins, Ann. Allergy Asthma Immunol.74: 279-283.

A “therapeutically effective dosage” for tumor therapy can be measuredby objective tumor responses which can either be complete or partial. Acomplete response (CR) is defined as no clinical, radiological or otherevidence of disease. A partial response (PR) results from a reduction inaggregate tumor size of greater than 50%. Median time to progression isa measure that characterizes the durability of the objective tumorresponse.

A “therapeutically effective dosage” for tumor therapy can also bemeasured by its ability to stabilize the progression of disease. Theability of a compound to inhibit cancer can be evaluated in an animalmodel system predictive of efficacy in human tumors. Alternatively, thisproperty of a composition can be evaluated by examining the ability ofthe compound to inhibit cell growth or apoptosis by in vitro assaysknown to the skilled practitioner. A therapeutically effective amount ofa therapeutic compound can decrease tumor size, or otherwise amelioratesymptoms in a subject. One of ordinary skill in the art would be able todetermine such amounts based on such factors as the subject's size, theseverity of the subject's symptoms, and the particular composition orroute of administration selected.

The composition must be sterile and fluid to the extent that thecomposition is deliverable by syringe. In addition to water, the carriercan be an isotonic buffered saline solution, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyetheylene glycol,and the like); and suitable mixtures thereof. Proper fluidity can bemaintained, for example, by use of coating such as lecithin, bymaintenance of required particle size in the case of dispersion and byuse of surfactants. In many cases, it is preferable to include isotonicagents, for example, sugars, polyalcohols such as mannitol or sorbitol,and sodium chloride in the composition. Long-term absorption of theinjectable compositions can be brought about by including in thecomposition an agent which delays absorption, for example, aluminummonostearate or gelatin.

When the active compound is suitably protected, as described above, thecompound may be orally administered, for example, with an inert diluentor an assimilable edible carrier.

IV. Uses and Methods of the Invention

The antibodies (including immunoconjugates, bispecifics/multispecifics,compositions and other derivatives described herein) of the presentinvention have numerous therapeutic utilities involving the treatment ofdisorders involving cells expressing CLD18. For example, the antibodiescan be administered to cells in culture, e.g., in vitro or ex vivo, orto human subjects, e.g., in vivo, to treat or prevent a variety ofdisorders such as those described herein. As used herein, the term“subject” is intended to include human and non-human animals whichrespond to the antibodies against CLD18. Preferred subjects includehuman patients having disorders that can be corrected or ameliorated bykilling diseased cells, in particular cells characterized by an alteredexpression pattern of CLD18 compared to normal cells.

A therapeutic effect in the treatments discussed herein is preferablyachieved through the functional properties of the antibodies of theinvention to mediate killing of cells e.g. by inducing complementdependent cytotoxicity (CDC) mediated lysis, antibody dependent cellularcytotoxicity (ADCC) mediated lysis, apoptosis, homotypic adhesion,and/or phagocytosis, preferably by inducing CDC mediated lysis and/orADCC mediated lysis.

For example, in one embodiment, antibodies of the present invention canbe used to treat a subject with a tumorigenic disorder, e.g., a disordercharacterized by the presence of tumor cells expressing CLD18 including,for example, gastric cancer. Examples of tumorigenic diseases which canbe treated and/or prevented encompass all CLD18 expressing cancers andtumor entities including stomach cancer, esophageal cancer, pancreaticcancer, long cancer, ovarian cancer, breast cancer, colorectal cancer,hepatic cancer, cancer of the gallbladder and head-neck cancer. Thesecancers may be in early, intermediate or advanced stages, e.g.metastasis.

The pharmaceutical compositions and methods of treatment describedaccording to the invention may also be used for immunization orvaccination to prevent a disease described herein.

In another embodiment, antibodies of the invention can be used to detectlevels of CLD18 or particular forms of CLD18, or levels of cells whichcontain CLD18 on their membrane surface, which levels can then be linkedto certain diseases or disease symptoms such as described above.Alternatively, the antibodies can be used to deplete or interact withthe function of CLD18 expressing cells, thereby implicating these cellsas important mediators of the disease. This can be achieved bycontacting a sample and a control sample with the anti-CLD18 antibodyunder conditions that allow for the formation of a complex between theantibody and CLD18. Any complexes formed between the antibody and CLD18are detected and compared in the sample and a control sample, i.e. areference sample.

Antibodies of the invention can be initially tested for their bindingactivity associated with therapeutic or diagnostic uses in vitro. Forexample, the antibodies can be tested using flow cytometric assays asdescribed herein.

Moreover, activity of the antibodies in triggering at least oneeffector-mediated effector cell activity, including inhibiting thegrowth of and/or killing of cells expressing CLD18, can be assayed. Forexample, the ability of the antibodies to trigger CDC and/or apoptosiscan be assayed. Protocols for assaying for CDC, homotypic adhesion,molecular clustering or apoptosis are described herein.

The antibodies of the invention can be used to elicit in vivo or invitro one or more of the following biological activities: to inhibit thegrowth of and/or differentiation of a cell expressing CLD18; to kill acell expressing CLD18; to mediate phagocytosis or ADCC of a cellexpressing CLD18 in the presence of effector cells; to mediate CDC of acell expressing CLD18 in the presence of complement; to mediateapoptosis of a cell expressing CLD18; to induce homotypic adhesion;and/or to induce translocation into lipid rafts upon binding CLD1 8.

In a particular embodiment, the antibodies are used in vivo or in vitroto treat, prevent or diagnose a variety of CLD18-related diseases.Examples of CLD18-related diseases include, among others, cancers suchas gastric cancer, pancreatic cancer, esophageal cancer, lung cancer andcancers as those listed above.

CLD18A2 is also expressed in differentiated normal stomach cells.Possible antibody induced clinical side effects by killing of thesecells may be reduced or avoided by parallel administration of stomachprotective drugs such as antacid, or inhibitors of the gastric protonpump such as omeprazol or related drugs.

Suitable routes of administering the antibody compositions of theinvention in vivo and in vitro are well known in the art and can beselected by those of ordinary skill.

As described above, anti-CLD18 antibodies of the invention can beco-administered with one or other more therapeutic agents, e.g., acytotoxic agent, a radiotoxic agent, antiangiogenic agent or andimmunosuppressive agent to reduce the induction of immune responsesagainst the antibodies of invention. The antibody can be linked to theagent (as an immunocomplex) or can be administered separate from theagent. In the latter case (separate administration), the antibody can beadministered before, after or concurrently with the agent or can beco-administered with other known therapies, e.g., an anti-cancertherapy, e.g., radiation. Such therapeutic agents include, among others,anti-neoplastic agents such as listed above. Co-administration of theanti-CLD18 antibodies of the present invention with chemotherapeuticagents provides two anti-cancer agents which operate via differentmechanisms yielding a cytotoxic effect to tumor cells. Suchco-administration can solve problems due to development of resistance todrugs or a change in the antigenicity of the tumor cells which wouldrender them unreactive with the antibody.

In another particular embodiment of the invention, the subject beingadministered the antibody is additionally treated with an antigenicagent including antibodies targeting VEGF or VEGFR and one or morechemical compounds inhibiting angiogenesis. Pretreatment with orparallel application of these drugs may improve the penetration ofantibodies in bulk tumors.

In another particular embodiment of the invention, the subject beingadministered the antibody is additionally treated with a compoundinhibiting growth factor receptor signaling including monoclonalantibodies binding to the EGFR receptor as well as chemical compoundsinhibiting signaling initiated by the EGFR, Her1 or Her2/neu receptor.

Target-specific effector cells, e.g., effector cells linked tocompositions (e.g. antibodies, multispecific and bispecific molecules)of the invention can also be used as therapeutic agents. Effector cellsfor targeting can be human leukocytes such as macrophages, neutrophilsor monocytes. Other cells include eosinophils, natural killer cells andother IgG- or IgA-receptor bearing cells. If desired, effector cells canbe obtained from the subject to be treated. The target-specific effectorcells can be administered as a suspension of cells in a physiologicallyacceptable solution. The number of cells administered can be in theorder of 10⁸ to 10⁹ but will vary depending on the therapeutic purpose.In general, the amount will be sufficient to obtain localization at thetarget cell, e.g., a tumor cell expressing CLD18, and to effect cellkilling by, e.g., phagocytosis. Routes of administration can also vary.

Therapy with target-specific effector cells can be performed inconjunction with other techniques for removal of targeted cells. Forexample, anti-tumor therapy using the compositions of the inventionand/or effector cells armed with these compositions can be used inconjunction with chemotherapy. Additionally, combination immunotherapymay be used to direct two distinct cytotoxic effector populations towardtumor cell rejection. For example, anti-CLD18 antibodies linked toanti-Fc-RI or anti-CD3 may be used in conjunction with IgG- orIgA-receptor specific binding agents.

Bispecific and multispecific molecules of the invention can also be usedto modulate Fc-gammaR or Fc-alphaR levels on effector cells, such as bycapping and eliminating receptors on the cell surface. Mixtures ofanti-Fc receptors can also be used for this purpose.

The compositions (e.g., antibodies, multispecific and bispecificmolecules and immunoconjugates) of the invention which have complementbinding sites, such as portions from IgG1, -2, or -3 or IgM which bindcomplement, can also be used in the presence of complement. In oneembodiment, ex vivo treatment of a population of cells comprising targetcells with a binding agent of the invention and appropriate effectorcells can be supplemented by the addition of complement or serumcontaining complement. Phagocytosis of target cells coated with abinding agent of the invention can be improved by binding of complementproteins. In another embodiment target cells coated with thecompositions of the invention can also be lysed by complement In yetanother embodiment, the compositions of the invention do not activatecomplement.

The compositions of the invention can also be administered together withcomplement. Accordingly, within the scope of the invention arecompositions comprising antibodies, multispecific or bispecificmolecules and serum or complement. These compositions are advantageousin that the complement is located in close proximity to the antibodies,multispecific or bispecific molecules.

Alternatively, the antibodies, multispecific or bispecific molecules ofthe invention and the complement or serum can be administeredseparately. Binding of the compositions of the present invention totarget cells causes translocation of the CLD18 antigen-antibody complexinto lipid rafts of the cell membrane. Such translocation creates a highdensity of antigen-antibody complexes which may efficiently activateand/or enhance CDC.

Also within the scope of the present invention are kits comprising theantibody compositions of the invention (e.g., antibodies andimmunoconjugates) and instructions for use. The kit can further containone or more additional reagents, such as an immunosuppressive reagent, acytotoxic agent or a radiotoxic agent, or one or more additionalantibodies of the invention (e.g., an antibody having a complementaryactivity).

Accordingly, patients treated with antibody compositions of theinvention can be additionally administered (prior to, simultaneouslywith, or following administration of a antibody of the invention) withanother therapeutic agent, such as a cytotoxic or radiotoxic agent,which enhances or augments the therapeutic effect of the antibodies ofthe invention.

In other embodiments, the subject can be additionally treated with anagent that modulates, e.g., enhances or inhibits, the expression oractivity of Fc-gamma or Fc-alpha receptors by, for example, treating thesubject with a cytokine. Preferred cytokines include granulocytecolony-stimulating factor (G-CSF), granulocyte-macrophagecolony-stimulating factor (GM-CSF), interferon-γ (IFN-γ), and tumornecrosis factor (TNF). Other important agents for increasing thetherapeutic efficacy of the antibodies and pharmaceutical compositionsdescribed herein are β-glucans which are homopolysaccharides of branchedglucose residues and are produced by a variety of plants andmicroorganisms, for example, bacteria, algae, fungi, yeast and grainsFragments of β-glucans produced by organisms may be also be used.Preferably, the β-glucan is a polymer of β(1,3) glucose wherein at leastsome of the backbone glucose units, e.g. 3-6% of the backbone glucoseunits, possess branches such as β(1,6) branches.

In a particular embodiment, the invention provides methods for detectingthe presence of CLD18 antigen in a sample, or measuring the amount ofCLD18 antigen, comprising contacting the sample, and a control sample,with a antibody which specifically binds to CLD18, under conditions thatallow for formation of a complex between the antibody or portion thereofand CLD18. The formation of a complex is then detected, wherein adifference complex formation between the sample compared to the controlsample is indicative for the presence of CLD18 antigen in the sample.

In still another embodiment, the invention provides a method fordetecting the presence or quantifying the amount of CLD18-expressingcells in vivo or in vitro.

The method comprises (i) administering to a subject a composition of theinvention conjugated to a detectable marker; (ii) exposing the subjectto a means for detecting said detectable marker to identify areascontaining CLD18-expressing cells.

Methods as described above are useful, in particular, for diagnosingCLD18-related diseases and/or the localization of CLD18-related diseasessuch as cancer diseases. Preferably an amount of CLD18, preferablyCLD18-A2 in a sample which is higher than the amount of CLD18,preferably CLD18-A2, in a control sample is indicative for the presenceof a CLD18-related disease in a subject, in particular a human, fromwhich the sample is derived.

In yet another embodiment immunoconjugates of the invention can be usedto target compounds (e.g., therapeutic agents, labels, cytotoxins,radiotoxins immunosuppressants, etc.) to cells which have CLD18expressed on their surface by linking such compounds to the antibody.Thus, the invention also provides methods for localizing ex vivo or invitro cells expressing CLD18, such as circulating tumor cells.

The present invention is further illustrated by the following exampleswhich are not to be construed as limiting the scope of the invention.

EXAMPLES 1. Generation of Murine Antibodies Against CLD18

a. Immunizations:

Balb/c or C57/BL6 mice were immunized with eucaryotic expressionvectors, encoding human CLD18 fragments (SEQ NO: 15, 16; 17, 18). 50 μgor 25 μg of plasmid DNA was injected into the quadriceps (intramuscular,i.m.) on days 1 and 10 for generation of monoclonal antibodies of Set1or alternatively on days 1 and 9, 1 and 11, or 1, 16 and 36 forgeneration of monoclonal antibodies of Set2 in the presence ofadjuvants, for example CpG (for details see Tab. 1b). CpG as well ascells transfected with CLD18A2 (SEQ ID NO: 1) alone or co-transfectedadditionally with murine soluble CD40L encoding RNA were injectedintramuscularly, PEI-Man was injected intramuscularly orintraperitoneally. The presence of antibodies directed against humanCLD18 in sera of mice was monitored by immune fluorescence microscopybetween day 16 and 43 depending on the specific immunization protocolused. The immune fluorescence was determined using HEK293 cellstransiently transfected with a nucleic acid encoding a fusion constructcomprising human CLD18A2 (SEQ ID NOs: 1, 2) and a fluorescent reporterprotein. Mice with detectable immune responses (FIG. 1) were boostedthree days prior to splenectomy for generation of monoclonal antibodiesof Set1, or mice were boosted three days, three and two days, or micewere boosted four, three and two days prior to splenectomy forgeneration of monoclonal antibodies of Set2 by intraperitoneal injectionof 5×10⁷ or alternatively 1×10⁸ HEK293 cells transiently transfectedwith a nucleic acid encoding human CLD18A2 (SEQ ID NOs: 1, 2) (fordetails see Tab. 1b). In Tab. 1a the immunization protocols used arededicated to the respective monoclonal antibodies.

TABLE 1a Immunisation protocols used for generation of monoclonalantibodies Immunisation Immunisation mAB protocol¹ mAB protocol Set124H5 40 42E12 45 26B5 40 43A11 45 26D12 40 44E10 45 28D10 40 47D12 4537G11 45 61C2 45 37H8 45 75B8  6 38G5 45 85A3  6 38H3 45 9E8 40 39F11 4519B9 40 41C6 45 Set2 45C1 53 166E2 51 125E1 45 175D10 51 163E12 51 ¹Forspecific immunization protocols see Tab. 1b

TABLE 1b Detailed immunisation protocols Immunisation Boosts withtransfected cells (prime and boosts with DNA) Cells co-transfected withDNA Cells with CLD18A2 vectors transfected (SEQ ID NO: 1) Immuni-encoding Serum- with CLD18A2 and with murine sation CLD18 withmonitoring (SEQ ID NO: 1) soluble CD40L days prior to protocol fragmentsadjuvant on day on day alone encoding RNA splenectomy 6 SEQ ID NO: 15:50 μg CpG 1 and 18 5 × 10⁷ transfected none 3 50 μg 10 MC3T3 cells 40SEQ ID NO: 17: 50 μg CpG 1 and 18 5 × 10⁷ HEK293 3 50 μg 10 cells; 100μg CPG as adjuvant 45 SEQ ID NO: 15: 50 μg CpG 1 and 16 1 × 10⁸ HEK293 350 μg 9 cells 51 SEQ ID NO: 15: 2.5 μl PEI- 1, 16 22, 30 5 × 10⁷transfected none 3 and 2 25 μg Man² and 36 and 43 HEK293 cells (150 mM)in H₂O with 5% Glucose 53 Priming: SEQ ID 50 μg CpG 1 and 20 5 x 10⁷transfected none 4, 3 and 2 NO: 15: 25 μg, in in H₂O 11 HEK293 cells andSEQ ID with 5% NO: 17: 25 μg; Glucose Boosting: SEQ ID NO: 17: 50 μg ²Invivo-jetPEI ™-Man from PolyPlus Transfection

b. Generation of Hybridomas Producing Human Monoclonal Antibodies toCLD18:

Mouse splenocytes were isolated and fused with PEG to a mouse myelomacell line based on standard protocols. The resulting hybridomas werethen screened for production of immunoglobulins with CLD18 specificityusing HEK293 cells transfected with a nucleic acid encoding human CLD18by FACS analysis. Single cell suspensions of splenic lymphocytes fromimmunized mice were fused with P3X63Ag8U.1 nonsecreting mouse myelomacells (ATCC, CRL 1597) in a 2:1 ratio using 50% PEG (Roche Diagnostics,CRL 738641). Cells were plated at approximately 3×10⁴/well in flatbottom microtiter plates, followed by about two week incubation inselective medium containing 10% fetal bovine serum, 2% hybridoma fusionand cloning supplement (HFCS, Roche Diagnostics, CRL 1 363 735) plus 10mM HEPES, 0.055 mM 2-mercaptoethanol, 50 μg/ml gentamycin and 1×HAT(Sigma, CRL H0262). After 10 to 14 days individual wells were screenedby flow cytometry for anti-CLD18 monoclonal antibodies. The antibodysecreting hybridomas were replated, screened again and, if stillpositive for anti-CLD18 monoclonal antibodies, were subcloned bylimiting dilution. The stable subclones were then cultured in vitro togenerate small amounts of antibody in tissue culture medium forcharacterization. At least one clone from each hybridoma, which retainedthe reactivity of parent cells (by FACS), was chosen. 9 vial cell bankswere generated for each clone and stored in liquid nitrogen.

c. Selection of Monoclonal Antibodies Binding to CLD18:

To determine the isotype of antibodies, an isotype ELISA was performed.The mouse monoAB ID Kit (Zymed, CRL 90-6550) or alternatively theIsoStrip Mouse Monoclonal Antibody Isotyping Kit (Roche, Cat. No.1493027) was used to determine Ig subclasses of the identified CLD18reactive monoclonal antibodies. Defined as Set1, nineteen hybridoma celllines were generated, six from a fusion of cells from a C57/BL6 mouseimmunized with CLD18A2-LoopD3 (SEQ ID NOs: 17, 18), thirteen from afusion of cells from a Balb/c mouse immunized with CLD18A2-Loop1 (SEQ IDNOs: 15, 16), expressing the following antibodies:

24H5, 26B5, 26D12, 28D10, 37G11, 37H8, 38G5, 38H3, 39F11, 4106, 42E12,43A11, 44E10, 47D12, 61C2, 75B8, 85A3, 9E8, 19B9

24H5: mouse monoclonal IgG2b, κ antibody, 182-D758-034

26B5: mouse monoclonal IgG2a, κ antibody, 182-D758-035, DSM ACC2745

26D12: mouse monoclonal IgG3, κ antibody, 182-D758-036, DSM ACC2746

28D10: mouse monoclonal IgG3, κ antibody, 182-D758-040, DSM ACC2747

37G11: mouse monoclonal IgG2a, κ antibody, 182-D1106-055, DSM ACC2737

37H8: mouse monoclonal IgG3, κ antibody, 182-D1106-056, DSM ACC2738

38G5: mouse monoclonal IgG3, κ antibody, 182-D1106-057, DSM ACC2739

38H3: mouse monoclonal IgG3, κ antibody, 182-D1106-058, DSM ACC2740

39F11: mouse monoclonal IgG3, κ antibody, 182-D1106-059, DSM ACC2741

41C6: mouse monoclonal IgG2a, κ antibody, 1$2-D1106-060

42E12: mouse monoclonal IgG2a, κ antibody, 182-D1106-061, DSM ACC2748

43A11: mouse monoclonal IgG2a, κ antibody, 182-D1106-062, DSM ACC2742

44E10: mouse monoclonal IgG3, κ antibody, 182-D1106-063

47D12: mouse monoclonal IgG3, κ antibody, 182-D1106-064

61C2: mouse monoclonal IgG2b, κ antibody, 182-D1106-067, DSM ACC2743

75B8: mouse monoclonal IgM, κ antibody, 182-D756-001

85A3: mouse monoclonal IgM, κ antibody, 182-D756-002

9E8: mouse monoclonal IgM, κ antibody, 182-D758-011

19B9: mouse monoclonal IgM, κ antibody, 182-D758-024

Defined as Set2, five hybridoma cell lines were generated, one from afusion of cells from a Balb/c mouse immunized with CLD18A2-LoopD3 (SEQID NOs: 17, 18) and CLD18A2-LoopD1 (SEQ ID NOs: 15, 16), four from afusion of cells from a Balb/c mouse immunized with CLD18A2-LoopD1 (SEQNOs: 15, 16), expressing the following antibodies:

45C1, 125E1, 163E12, 166E2, 175D10

45C1: mouse monoclonal IgG2a, κ antibody, 182-D758-187

125E1: mouse monoclonal IgG2a, κ antibody, 182-D1106-279, DSM ACC2808

163E12: mouse monoclonal IgG3, κ antibody, 182-D1106-294, DSM ACC2809

166E2: mouse monoclonal IgG3, κ antibody, 182-D1106-308

175D10: mouse monoclonal IgG1, κ antibody, 182-D1106-362, DSM ACC2810

2. Production of Monoclonal Antibodies

Production and Purification of Monoclonal Antibodies Reactive to CLD18:

To produce mg amounts of antibody for functional characterization,hybridoma cells were seeded in dialysis based bioreactors (CELLineCL1000, Integra, Chur, CH) at 2×10⁶ cells/ml. Antibody containingsupernatant was harvested once weekly. Mouse monoclonal antibody waspurified using Melon Gel (Pierce, Rockford, USA) and concentrated byammonium sulphate precipitation or alternatively purified by ProteinAusing FPLC. Antibody concentration and purity was determined byBCA-Assay and purity checked by sodium dodecylsulphate gelelectrophoresis and coomassie staining.

3. Binding Characteristics of Monoclonal Antibodies

a. Quality Control of Transfectants in WB, IF:

To generate CLD18A2 expressing cells, HEK293 or CHO cells weretransfected with nucleic acids encoding CLD18A2 (SEQ ID NOs: 1, 2) orCLD18A2-myc (SEQ ID NOs: 3, 4).

HEK293 cells were transfected with CLDN18A2-myc (SEQ ID NOs: 3, 4) orleft untransfected. 24 hours post transfection, cells were harvested,lysed and subjected to sodium dodecylsulphate gel electrophoresis. Thegel was blotted and stained with a mouse anti-myc antibody. Afterincubation with a peroxidase labelled anti mouse antibody, the blot wasdeveloped with ECL reagent and visualized using a LAS-3000 imager(Fuji). Only in the transfected cells but not in the negative control, aband with the expected molecular weight of CLD18-myc was observed (FIG.2).

CHO cells were transfected with CLD18A2 (SEQ ID NOs: 1, 2) and grown onchamber slides for 24 h. Cells were fixed with methanol and stained witha rabbit polyclonal antibody against CLD18 at 1 μg/ml for 60 min. at 25°C. After washing, cells were stained with an Alexa488 labelled goatanti-rabbit IgG (Molecular Probes) and evaluated by fluorescencemicroscopy. FIG. 3 shows transfected CHO cells, expressing CLD18 on thecell membrane as well as untransfected cells. These heterologously CLD18expressing cells were used for the following assays to test thespecificity of antibody binding.

b. Selection of Monoclonal Antibodies Binding to CLD18/Primary Screensby Flow Cytometry:

HEK293 cells were co-transfected with expression vectors encoding humanCLD18A2 (SEQ ID NOs: 1, 2) and a fluorescing reporter protein 40 h priorto the assay or alternatively HEK293 cells stably expressing humanCLD18A2 (HEK293-CLD18A2) were used and counterstained with propidiumiodide (PI). After cell detachment using 2 mM EDTA/PBS cells were washedwith complete growth medium and plated at approximately 1-5×10⁵cells/well in U-bottom microtiter plates. Cells were incubated for 30min. at 4° C. with hybridoma supernatant followed by two washing stepswith 1% heat inactivated FBS/PBS and finally incubation with APC orAlexa647-conjugated anti-mouse IgG specific secondary antibody. Aftertwo washing steps, co-transfected cells were fixed with CellFIX (BDBiosciences). Binding was assessed by flow cytometry using a BDFACSArray. Fluorescence marker expression is plotted on the horizontalaxis against antibody binding on the vertical axis. All mouse antibodies24H5, 26B5, 26D12, 28D10, 37G11, 37H8, 38G5, 38H3, 39F11, 4106, 42E12,43A11, 44E10, 47D12, 61C2, 75B8, 85A3, 9E8, 19B9, 45C1, 125E1, 163E12,166E2, and 175D10 were detected to bind specifically to the surface offluorescence marker expressing cells (FIG. 4, cells in Q2) asexemplified for hybridoma supernatants containing monoclonal antibodies24H5 (FIG. 4A, cells in Q2), 85A3 (FIG. 4B), 175D10, 125E1, 163E12,166E2 and 45C1 (FIG. 4C, cells in Q1).

c. Comparison of Antibody Binding to Myc- or HA-Tagged CLD18A2:

The binding characteristics of the identified CLD18-specific monoclonalantibodies were further specified. Therefore, monoclonal antibodybinding was analyzed to CLD mutants, created by insertion of epitopetags. CLD18A2-HA (SEQ ID NO: 6) contains a HA-epitope tag inCLD18A2-loop1, whereas CLD18A2-Myc (SEQ ID NO: 4) contains a Myc-epitopetag inserted into CLD18A2-loop2. As insertion of these tags causesdestruction of epitopes, the identified monoclonal antibodies, can begrouped according to the loss of binding to any of the mutants. HEK293cells transiently co-transfected with a fluorescence marker and humanCLD18A2, or with a fluorescence marker and CLD18A2-HA, or with afluorescence marker and CLD18A2-Myc were incubated with hybridomasupernatants containing CLD18-specific monoclonal antibodies for 30 min.at 4° C., followed by incubation with Alexa647-conjugated anti-mouse IgGsecondary antibody. Before analysis on a BD FACSArray, cells were fixedusing CellFIX. As exemplified for 24H5, 9E8, 26B5 and 19B9 in FIG. 5,monoclonal antibodies could be separated based on their bindingcharacteristics into four different groups: (i) antibodies that bind tounmodified CLD18A2 as well as to CLD18A2-HA and CLD18A2-Myc, e.g. 24H5,(FIG. 5A), or (ii) antibodies that do not bind to CLD18A2-HA, e.g. 9E8,(FIG. 5B), or (iii) antibodies that do not bind to CLD18A2-Myc, e.g.26B5, (FIG. 5C), or (iv) antibodies that do not bind to CLD18A2-HA norto CLD18A2-Myc, e.g. 19B9, (FIG. 5D).

d. Comparison of Antibody Binding to Human CLD18A1 Versus CLD18A2Transfectants by Flow Cytometry:

Binding specificity of the identified monoclonal antibodies to CLD18A2isoforms was analyzed by flow cytometry. HEK293 cells stably expressinghuman CLD18A2 (HEK293-CLD18A2) and HEK293 cells stably expressing humanCLD18A1 (SEQ ID NOs: 7, 8) (HEK293-CLD18A1) were incubated for 30 min.at 4° C. with hybridoma supernatants containing monoclonal antibodies,followed by incubation with Alexa647-conjugated anti-mouse IgG secondaryantibody and fixation of cells or alternatively without fixation butwith PI counterstaining. Binding was assessed by flow cytometry using aBD FACSArray. FIG. 6 shows examples for, the two groups of monoclonalantibodies that were identified in the panel comprised of 24H5, 26B5,26D12, 28D10, 37G11, 37H8, 38G5, 38H3, 39F11 4106, 42E12, 43A11, 44E10,47D12, 61C2, 75B8, 85A3, 9E8, 19B9, 45C1, 125E1, 163E12, 166E2, 175D10:(i) monoclonal antibodies 43A11, 45C1, and 163E12 bind specifically tohuman CLD18A2 but not to human CLD18A1 (FIG. 6A,B), and (ii) monoclonalantibody 37H8 binds to both human isoforms (FIG. 6A).

e. Comparison of Antibody Binding to Human CLD18A1 Versus CLD18A2Transfectants by Immunofluorescence Microscopy:

HEK293 cells were transiently transfected with an expression vectorencoding a fusion protein of CLD18A1 (SEQ ID NO: 8) or CLD18A2 (SEQ IDNO: 2) with a fluorescence reporter and grown on chamber slides. Cellswere either stained unfixed or after paraformaldehyde fixation withmonoclonal antibody containing tissue culture supernatant for 30 min. at37° C. After washing, cells were stained with an Alexa555-labelledanti-mouse Ig antibody (Molecular Probes). Binding of antibodies wasevaluated by fluorescence microscopy. As shown in FIG. 7, antibody 37G11specifically reacted with CLD18A2 (FIG. 7A) but not with CLD18A1 (FIG.7B). In contrast, antibody 26B5 was reactive with both, CLD18A2 andCLD18A1 (FIG. 8).

For antibodies 24H5, 26B5, 26D12, 28D10, 37G11, 37H8, 38G5, 38H3, 39F11,4106, 42E12, 43A11, 44E10, 47D12, 61C2, 75B8, 85A3, 9E8, 19B9, a cleardifference between staining of living cells and paraformaldehyde fixedcells was observed. The antibodies formed an uniform membrane stainingwhen cells were fixed (FIG. 7C, 8C, 8D). In contrast, incubation ofliving cells with these antibodies leads to the generation of proteinclusters, visible as a speckle like staining pattern (FIG. 7A, 8A, 8B).This shows that all antibodies bind to native epitopes as found on thesurface of living cells.

f. Determination of Endogenously Expressing Cell Lines:

A CLD18A2 gene-specific primer pair (SEQ ID NO: 11, 12) was used inRT-PCR analyses to screen cell lines for expression of CLD18A2. Humangastric carcinoma cell lines NCI-SNU-16 (ATCC CRL-5974), NUGC-4(JCRB0834) and KATO-III (ATCC HTB-103) and human pancreas adenocarcinomacell line DAN-G (DSMZ ACC249) were found to display robust endogenousexpression of CLD18 (FIG. 9). Expression was confirmed on protein levelby staining with a rabbit polyclonal serum against CLD18.

g. Staining of Endogenously Expressing Cell Lines with CLD18 SpecificAntibodies and Immunofluorescence Analysis:

DAN-G, SNU-16, NUGC-4 and KATO-III cells were grown on chamber slidesunder standard conditions. Cells were unfixed or alternatively fixedwith methanol and stained with the respective antibodies. For antibodies24H5, 26B5, 26D12, 28D10, 37G11, 37H8, 38G5, 38H3, 39F11, 4106, 42E12,43A11, 44E10, 47D12, 61C2, 75B8, 85A3, 9E8, 19B9 staining of the cellsurface was observed as exemplified in FIGS. 10, 11 and 12A. Forantibodies 45C1, 125E1, 163E12, 166E2, and 175D10 native epitoperecognition was assayed and cell surface staining was observed onunfixed cells as shown in FIG. 12B. Subgroups of antibodies showedhomogenous staining of the cell membrane either preponderantly atcell-cell interfaces or at free parts of the membrane not adjacent toother cells. Other antibodies stained discrete foci and aggregates onthe cell membrane altogether demonstrating that the respectiveantibodies bind to different epitopes including epitopes which aremasked by homotypic or heterotypic association of CLD18 as well as CLD18epitopes accessible in preformed tight junctions.

h. Staining of Endogenously Expressing Cell Lines by Flow Cytometry:

Surface expression of constitutively expressed CLD18A2 on KATO-III andNUGC-4 living cells was analyzed by flow cytometry. This is exemplifiedby KATO-III and NUGC-4 cells stained with monoclonal antibody 61C2 or163E12, followed by incubation with Alexa647-conjugated anti-mouse IgGsecondary antibody and fixation of cells or alternatively withoutfixation. Binding was assessed by flow cytometry using a BD FACSArray.FIG. 13 shows a strong binding of 61C2 to at least 70.3% of KATO-IIIcells and of 163E12 to CLD18A2 on KATO-III and NUGC-4 cells.

i. Sequence Alignment of Mouse and Human CLD18A1 and CLD18A2:

Human CLD18A2 (NP_001002026) and human CLD18A1 (NP_057453) in a sequencecomparison differ in the N-terminus and mouse CLD18 variants (NP_062789and AAL15636) demonstrate high homology and sequence variation sitesbetween the molecules (see FIG. 14).

j. Reactivity of Antibodies with Murine CLD18A1 and Murine CLD18A2Analyzed by flow cytometry:

Binding of the identified monoclonal antibodies to murine CLD18A2 andCLD18A1 was analyzed by flow cytometry. HEK293 cells transientlyco-transfected with a fluorescence marker and murine CLD18A2 (SEQ IDNOs: 33, 35) or with a fluorescence marker and murine CLD18A1 (SEQ IDNOs: 36, 37) were incubated with hybridoma supernatants containing thehuman CLD18-specific monoclonal antibodies 38G5, 38H3, 37G11, 45C1 and163E12, respectively, for 30 min. at 4° C., followed by incubation withAlexa647-conjugated anti-mouse IgG secondary antibody and fixation ofcells. Binding was assessed by flow cytometry using a BD FACSArray. FIG.15 shows three different binding profiles: 38G5, and 45C1 do not bind toany of the murine CLD18 isoforms, 37G11, and 163E12 bind to murineCLD18A2 but not to murine CLD18A1, and 38H3 binds to murine CLD18A1 andCLD18A2. These antibodies are valuable tools to determine a potentialtoxicity of CLD18 monoclonal antibodies in preclinical studies.

Altogether these data show, that monoclonal antibodies of the invention24H5, 26B5, 26D12, 28D10, 37G11, 37H8, 38G5, 38H3, 39F11, 4106, 42E12,43A11, 44E10, 47D12, 61C2, 75B8, 85A3, 9E8, 19B9, 45C1, 125E1, 163E12,166E2, and 175D10 generated against CLD18 represents a diversity ofbinding characteristics to different epitopes and topologies of humanCLD18.

A combination of different properties described in examples 3b, c, d, e,g, h, and j can be used to categorize monoclonal antibodies into suchdifferent classes.

4. Immunohistochemistry (IHC)

A CLD18A2 epitope specific antibody generated by immunization with thepeptide of SEQ ID NO: 21 was used for immunohistochemicalcharacterisation of CLD18A2 expression. Paraffin embedded tissuesections derived from a comprehensive panel of normal and tumor tissueswere used for protein expression and localisation analyses. Nosignificant expression was detected in any other normal organ tissueexcept stomach (see Tab. 2, FIG. 16A). In contrast, CLD18A2 expressionwas verified by, immunohistochemistry in different cancers includingstomach cancer and lung cancer (FIG. 16B).

Interestingly, expression of CLD18A2 protein in gastric mucosa wasrestricted to terminally differentiated cells of the gastric epitheliumin the base and pit regions. In contrast, cells in the neck region ofgastric mucosa, in particular gastric stem cells in the isthmus part,which replenish the entire mucosa, do not express CLD18A2 (FIG. 16C).

TABLE 2 CLD18A2 expression in normal and tumor tissues as analysed byIHC Tissue type Result Adrenal − Bladder − Blood cells − Bone Marrow −Breast − Colon − Endothelium − Esophagus − Fallopian tube − Heart −Kidney (glomerulus, tubule) − Liver − Lung − Lymph node − Ovary −Pancreas − Parathyroid − Pituitary − Placenta − Prostate − Skin − Spleen− Stomach + Striated muscle − Testis − Thymus − Thyroid − Ureter −Uterus (cervix, endometrium) −

The monoclonal antibody 39F11 was used for immunohistochemical CLD18A2specific studies. As shown in FIG. 17A, no significant reactivity wasdetectable on all tested normal tissues except stomach (FIG. 17A),whereas stomach carcinomas and lung carcinomas remain strongly positive(FIG. 17B).

Another group of antibodies of the invention shows a specific cancerstaining pattern with binding to stomach cancer but no reactivity withnormal stomach tissue. Such a staining pattern is shown in FIG. 18A withmonoclonal antibody 26B5.

Immunohistochemistry was used for specificity analysis of 175D10 (FIG.18B), 43A11 (FIG. 18C), 163E12 (FIG. 18D) and 45C1 (FIG. 18E) onsections derived from HEK293 tumor cell lines: HEK293 tumor cell linesstably expressing human CLD18A2 (HEK293-CLD18A2) or CLD18A1(HEK293-CLD18A1) or being transfected with an expression control plasmidcontaining only the antibiotic resistance gene for selection(HEK293-mock) were xenografted into mice to form solid tumors. Noexpression was detectable in mock-transfected HEK293 xenograft tumors.In contrast, strong and homogeneous membrane-staining was observed inHEK293-CLD18A2 xenograft tumors and in stomach carcinoma specimens.

5. Complement Dependent Cytotoxicity (CDC)

a. CDC of Monoclonal Antibodies of Sell as Measured by Flow Cytometry:

Plasma for complement lysis was prepared by drawing blood from healthyvolunteers into S-Monovette-EDTA vacutainer tubes (Sarstedt, Nürmbrecht,Germany) which were then centrifuged at 600 g for 20 min. Plasma washarvested and stored at −20° C.

In a first set of experiments hybridoma supernatants were analyzed fortheir capability to induce complement dependent cytotoxicity (CDC)against HEK293 cells stably expressing human CLD18A2 (HEK293-CLD18A2).Cells were incubated with hybridoma supernatants containing monoclonalantibodies 85A3, 28D10, 24H5 or 26D12, respectively for 20 min. at roomtemperature. Following centrifugation (5 min. at 450 g) the supernatantwas removed and 20% human plasma in DMEM (prewarmed to 37° C.) was addedto the cells and incubated for another 20 min. at 37° C. Thereafter,cell lysis was determined on FACS by using the propidium iodide (PI)staining method. PI was added to a final concentration of 2.5 μg/ml. Forflow cytometry, a BD FACSArray flow cytometer was used (BD Biosciences,Mountain View, Calif.). At least 10000 events were collected foranalysis with cell debris excluded by adjustment of the forward sidewardscatter (FCS) threshold. The percentage of lysed cells (PI-positivecells) is shown in FIG. 19. Monoclonal antibodies 85A3, 28D10 and 26D12induced lysis of 33.5%, 38.2% and 39.2%, respectively of HEK293-CLD18A2cells, whereas CDC mediated by 24H5 was only 19.3%.

b. CDC of Monoclonal Antibodies of Set1:

In a second set of experiments the specificity of monoclonal antibodiesto induce CDC on CLD expressing cells was analyzed. Therefore, a set ofantibodies binding either specific to human CLD18A2 or also binding tohuman CLD18A1 was tested for CDC-induction against CHO cells stablytransfected with human CLD18A2 (CHO-CLD18A2) or human CLD18A1(CHO-CLD18A1). CHO-CLD18A2 and CHO-CLD18A1 cells were seeded 24 h beforethe assay with a density of 3×10⁴/well in tissue-culture flat-bottommicrotiter plates. The next day growth medium was removed and the cellswere incubated in triplicates with hybridoma supernatants adjusted to aconcentration of 10 μg/ml containing monoclonal antibodies 24H5, 26D12,28D10, 37G11, 37H8, 38G5, 38H3, 39F11, 4106, 42E12, 43A11, 44E10, 47D12,and 61C2, respectively. Control cells were incubated with growth mediumor growth medium containing 0.2% saponin for the determination ofbackground lysis and maximal lysis, respectively. After incubation for20 min. at room temperature supernatant was removed and 20% human plasmain DMEM (prewarmed to 37° C.) was added to the cells and incubated foranother 20 min. at 37° C. Then, supernatants were replaced by PBScontaining 2.5 ethidium bromide and fluorescence emission afterexcitation at 520 nm was measured using a Tecan Safire. The percentagespecific lysis was calculated as follows: % specific lysis=(fluorescencesample−fluorescence background)/(fluorescence maximal lysis−fluorescencebackground)×100. FIG. 20 shows that monoclonal antibodies 26D12, 28D10,37H8, 38H3 and 39F11 mediate high, monoclonal antibody 38G5 mediatesmedium, monoclonal antibodies 4106 and 61C2 mediate low, and monoclonalantibodies 24H5, 37G11, 42E12, 43A11, 44E10 and 47D12 mediate no CDCagainst CHO-CLD18A2 cells. In contrast, none of the antibodies iscapable of inducing CDC against CHO-CLDA1 cells, although 26D12, 28D10,37H8, 38H3, 39F11, 4106, 47D12 and 61C2 also bind to CLD18A1 asdetermined by flow cytometry and immunofluorescence.

c. Monoclonal Antibody Titration and CDC Using Monoclonal Antibodies ofSet1:

To measure the ability of the anti-CLD18 antibodies to induce CDC at lowconcentrations, an experiment was performed where three differentantibodies were titrated. CHO-CLD18A2 cells growing in microtiter plateswere incubated with a concentration range of 75B8 (100, 30, 10, 3 and 1μg/ml), 37H8 (10, 3.3 and 1 μg/ml) and 28D10 (10, 1 and 0.1 μg/ml),respectively, for 20 min. at room temperature. Supernatant was removedand 20% human plasma in DMEM (prewarmed to 37° C.) was added to thecells and incubated for another 20 min. at 37° C. Before analysis usinga Tecan Safire, supernatants were replaced by PBS containing 2.5 μs/mlethidium bromide. FIGS. 21A-C show the percentage of specific lysis as afunction of antibody concentration. Monoclonal antibody 75B8 induceslysis of 31.0% CHO-CLD18A2 cells at 10 μs/ml, and drops to 6.2% at 1μg/ml (FIG. 21A), whereas monoclonal antibodies 28D10 and 37H8 stillinduce 39% and 26.5% specific lysis at 1 μg/ml (FIG. 21B, C),respectively.

d. CDC of Monoclonal Antibodies of Set2 as Measured by Flow Cytometry:

Serum for complement lysis was prepared by drawing blood from healthyvolunteers into Serum-Monovette vacutainer tubes (Sarstedt, Nürmbrecht,Germany) which were then centrifuged at 600 g for 20 min. Serum washarvested and stored at −20° C. Control serum was heat inactivated at56° C. for 30 min before storage.

Hybridoma supernatants were analyzed for their capability to inducecomplement dependent cytotoxicity (CDC) against KATO-III cellsendogenously expressing human CLD18A2. Cells were incubated with crudeor purified hybridoma supernatants containing monoclonal antibodies45C1, 125E1, 163E12, 166E2, and 175D10, respectively for 30 min. at 37°C. 20% human serum in RPMI was added to the cells and incubated foranother 30 min. at 37° C. Thereafter, cell lysis was determined on FACSby using the propidium iodide (PI) staining method. PI was added to afinal concentration of 2.5 μg/ml. For flow cytometry a BD FACSArray flowcytometer was used (BD Biosciences, Mountain View, Calif.). At least10000 events were collected for analysis with cell debris excluded byadjustment of the forward sideward scatter (FSC/SSC) threshold. Specificlysis was calculated by the following formula: specific lysis=(%PI-positive cells in sample−% PI-positive cells in sample with heatinactivated serum). Robust CDC mediated lysis was observed in particularfor 163E12.

6. Antibody-Dependent Cellular Cytotoxicity (ADCC)

Hybridoma supernatants were analyzed for their capability to induceantibody-dependent cellular cytotoxicity (ADCC) against HEK293 cellsstably expressing human CLD18A2 (HEK293-CLD18A2) or human CLD18A1(HEK293-CLD18A1).

a. Enrichment of human peripheral blood mononuclear cells: Human bloodfrom healthy donors was diluted twice in phosphate buffer (PBS) andblood cells were layered on Ficoll (Lymphocyte Separation Medium 1077g/ml, PAA Laboratories, cat. no. J15-004). Peripheral blood mononuclearcells (MNCs) were collected from the interphase, washed and resuspendedin RPMI 1640 culture medium supplemented with 10% heat-inactivated fetalcalf serum, 2 mM L-glutamine.

b. ADCC set up: Target cells were labeled with a fluorescence enhancingligand (BADTA, Perkin Elmer cytotoxicity assay kit DELFIA EuTDACytotoxicity Reagents, cat. no. AD0116) for 30 minutes. After extensivewashing in RPMI-10 supplemented with 10 mM probenecid (Sigma, cat. no.P8761), 10-20 mM HEPES, and 10% heat-inactivated fetal calf serum, thecells were adjusted to 1×10⁵ cells/ml. Labeled target cells, effectorcells (MNCs), and supernatants containing monoclonal antibodies adjustedto a concentration of 10 μg/ml were added to round-bottom microtiterplates. For isolated effector cells, an effector to target (E:T) ratioof 100:1 (data not shown for 50:1 and 25:1) was used. After incubation(2 hours, 37° C.), assays were stopped by centrifugation, andfluorescence ligand release from duplicates was measured in europiumcounts in a time-resolved fluorometer. Percentage of cellularcytotoxicity was calculated using the following formula: % specificlysis=(experimental release counts−spontaneous release counts)/(maximalrelease counts−spontaneous release counts)×100, with maximalfluorescence ligand release determined by adding Triton X-100 (0.25%final concentration) to target cells, and spontaneous release measuredin the absence of antibodies and effector cells. FIG. 22 shows thatmonoclonal antibodies 26B5, 37H8, 38G5, 47D12, and 61C2 mediate ADCCagainst HEK293-CLD18A2 cells. In contrast, these antibodies induce nosignificant or only low level cytotoxicity on CLD18A1 targetsdemonstrating a CLD18A2 specific ADCC (FIG. 23).

7. Proliferation Inhibition

Purified murine monoclonal antibodies were analyzed for their capabilityto inhibit cell growth of KATO-III cells endogenously expressing humanCLD18A2.

1×10⁴ target cells endogenously expressing CLD18A2 (KATO-III) werecultured in the presence of approximately 10 μg monoclonal antibodies.

DELFIA Cell Proliferation Kit (Perkin-Elmer, Cat. No. AD0200) is anon-isotopic immunoassay based on the measurement of5-bromo-2′-deoxyuridine (BrdU) incorporation during DNA synthesis ofproliferating cells in microplates. Incorporated BrdU is detected usingeuropium labelled monoclonal antibody. To allow antibody detection cellsare fixed and DNA denatured using Fix solution. Unbound antibody iswashed away and DELFIA inducer is added to dissociate europium ions fromthe labelled antibody into solution, where they form highly fluorescentchelates with components of the DELFIA Inducer. The fluorescencemeasured—utilizing time-resolved fluorometry in the detection—isproportional to the DNA synthesis in the cell of each well.

Strong inhibition of proliferation was observed with antibodies 125E1,163E12, 45C1, 37G11, 371H8, 28D10 and 166E2, respectively. Moderateinhibition of proliferation was observed with murine antibodies 43A11,175D10, 42E12, 26D12, 61C2 and 38H3, respectively.

8. Performance in Therapeutic Mouse Xenograft Models

Therapeutic potential of the identified monoclonal antibodies bindingspecifically to CLD18A2 was studied in therapeutic xenograft models.

a. Early Treatment of Highly CLD18A2 Expressing Tumors in Mice

SCID mice were subcutaneously inoculated with 1×10⁷ HEK293 cells stablyexpressing high levels of human CLD18A2 (HEK293-CLD18A2). Expressionlevels of human CLD18A2 in HEK293-CLD18A2 cells were comparable withexpression levels in primary gastric cancers from patients. Eachexperimental treatment group comprised 10 mice (number of mice per groupn=10). Therapy of mice started 3 days after tumor inoculation. 200 μs ofpurified hybridoma supernatants representing murine monoclonalantibodies 26B5, 26D12, 28D10, 37G11, 37H8, 38G5, 39F11, 42E12, 43A11,38H3, or 61C2 were injected once per week for 4 weeks intravenously.Alternatively 200 μg of purified hybridoma supernatants containingmurine monoclonal antibodies 45C1, 125E1, 163E12, 166E2, or 175D10 wereadministered twice per week for 6 weeks by alternating intravenous andintraperitoneal injection. Tumor growth of treated mice was monitoredtwice per week (Tumor Volume=Length×Width×Width divided by 2 in mm³).The mice were killed if the tumor reached a volume of 500 mm³ or in caseof severe morbidity. FIG. 24 exemplifies robust inhibition ofHEK293-CLD18A2 tumor cell growth by antibodies of the invention. FIGS.25A and 25B show prolongation of survival by treatment with antibodiesof the invention in an early treatment xenograft model usingHEK293-CLD18A2 cells.

b. Late Onset Treatment of Advanced Highly CLD18A2 Expressing Tumors inMice

The same tumor xenograft model based on HEK293-CLD18A2 cells wasdesigned as a late therapy onset protocol as opposed to the earlytreatment described above. On day 27 after tumor cell inoculation micewere randomized in test groups each comprising 5-6 mice and therapy wasinitiated with 200 μg of purified hybridoma supernatants containingmurine monoclonal antibodies 43A11, 163E12, and 175D10, respectively.Antibodies were administered twice per week for 6 weeks by alternatingintravenous and intraperitoneal injection. Also in this model antibodiesof the invention were shown to inhibit tumor growth. For severalantibodies this resulted in prolongation of survival (FIG. 26).

c. Early Treatment of Tumors Expressing Low Levels of CLD18A2

SCID mice were subcutaneously inoculated with 2×10⁵ cells of the DAN-Gtumor cell line, an infiltrating human pancreatic adenocarcinoma cellline that constitutively expresses CLD18A2 protein at low level.Treatment of mice (10 per group) was initiated 3 days after tumorgrafting: 200 μg of purified hybridoma supernatants containing murinemonoclonal antibodies 45C1, 125E1, 163E12, 166E2, or 175D10 wereadministered twice per week for 6 weeks by alternating intravenous andintraperitoneal injection. Owing to the aggressive and fast tumor growthof the pancreatic DAN-G tumor cell line in vivo mice developed tumorcachexia and died within a few days. Even though, as a consequence, thewindow for measuring therapeutic effects was narrow, tumor growthinhibition and prolonged survival mediated by antibodies of theinvention was also observed in this model (FIGS. 27A and 27B).

d. Antibodies of the Invention do not Elicit Side Effects in Mice

A murine CLD18A2-specific primer pair (s: CTA CCA AGO GCT ATG GCG TTC,as: GCA CCG AAG GTG TAC CTG GTC) was used in RT-PCR analyses to amplifycDNA derived from a comprehensive panel of normal mouse tissues (seeFIG. 28).

Expression of murine CLD18A2 was not detectable in any tested normaltissues, except stomach (see FIG. 28). Furthermore, an CLD18A2 specificantibody, which crossreacts with human and mouse CLD18A2, was used forimmunohistochemical analysis of CLD18A2 expression in a large panel ofnormal mouse tissues (see Tab. 3). Except for normal gastric tissue alltested normal tissues show no CLD18A2 expression. As we observed for thehuman CLD18A2, we also found for the mouse counterpart that while thesurface epithelia- and deeper crypt cells express CLD18A2 at their cellsurface, the central neck region is CLD18A2 negative (see FIG. 29 A-C).In summary, tissue distribution of CLD18A2 appears to be identical inmen and mice.

TABLE 3 CLD18 expression within murine normal tissues as analysed byimmunhistochemistry tissue CLD18 expression cerebellum − cerebrum −colon − esophagus − heart − kidney − liver − lung − lymph node − ovary −pancreas − skeletal muscle − spleen − stomach + thymus − bladder −

We further investigated potential side effects mediated by antibodies125E1, 163E12, 166E2 and 175D10 in mice. All of these antibodies hadbeen previously shown by FACS analysis to react with the murine CLD18A2as well as with the human protein.

Neither were any visible side effects observed in mice during and aftertreatment with these antibodies, nor were any histomorphologicalcorrelates of toxicity observed in the gastric mucosa of antibodytreated mice as compared to untreated (PBS-treated) mice (see FIG. 30).

9. Chimerization of Antibodies

a. Generation of Mouse/Human Chimeric Monoclonal Antibodies

Total RNA and subsequently single stranded cDNA was prepared from humanperipheral blood mononuclear cells (PBMC) and from human spleen tissueby standard methods known to those skilled in the art, for example byusing RNeasy Midi Kit (Qiagen) and Superscript II reverse transcriptase(Invitrogen).

The constant region of the human kappa light chain was amplified fromPBMC cDNA by PCR. The sense oligomer (SEQ ID NO:38) added a BamHIrestriction site at the 5′ end of the constant region and changed theoriginal nucleic acid sequence 5′-CGAACT-3′ coding for the first twoamino acids (Arg-Thr) of the constant region into 5′-CGTACG-3′,generating a BsiWI restriction site without changing the amino acidsequence. The antisense oligomer (SEQ ID NO:39) included a stop codonand added a NotI restriction site at the 3′ end of the amplifiedconstant region. The PCR product as well as a standard expression vector(for example pcDNA3.1(+), Invitrogen) were sequentially incubated withBamHI and NotI restriction enzymes. The vector was additionally treatedwith calf intestinal alkaline phosphatase to prevent recirculation. Theconstant region was finally ligated into the vector, so that anyforthcoming fusion of a variable region in front of the constant regionis now possible via a HindIII restriction site (5′-AAGCTT-3′) from theresidual vector multiple cloning site and via the BsiWI restriction site(5′-CGTACG-3′) generated with the PCR product. The sequence of the humankappa light chain constant region inserted into the vector is listed asSEQ ID NO:40, the amino acid sequence of the human kappa constant regionis listed as SEQ ID NO:41.

The constant region of the human gamma-1 heavy chain was amplified fromspleen cDNA by PCR. The 5′ phosphorylated sense oligomer (SEQ ID NO:42)was placed over the naturally occurring ApaI restriction site, located11 nucleotides downstream of the beginning of the constant region, andadded a HindIII restriction site at the 5′ end of the amplified part ofthe constant region. The 5′ phosphorylated antisense oligomer (SEQ IDNO: 43) included a stop codon and added a NotI restriction site at the3′ end of the thus amplified constant region. The thus generated PCRproduct was blunt ended and 5′ phosphorylated. The amplified gammaconstant region was verified to be of the IgG1 subclass by PCR with adiscriminating antisense oligomer (SEQ ID NO: 44) and by sequencing. Astandard expression vector (for example pcDNA3.1(+)(Hygro, Invitrogen)with a different antibiotic resistance (for example hygromycin) thanthat of the vector used for expression of the light chain (for exampleneomycin) was incubated with PmeI restriction enzyme to completelyremove the multiple cloning site leaving blunt ends. The vector wasadditionally treated with calf intestinal alkaline phosphatase toprevent recirculation. The constant region was finally ligated into thevector, so that any forthcoming fusion of a variable region in front ofthe constant region is now possible via the HindIII restriction site(5′-AAGCTT-3′) and via the ApaI restriction site (5′-GGGCCC-3′), bothgenerated with the PCR product. The correct orientation of the constantregion in the vector, i.e. suitable for the preceeding promoter of thevector, was verified by sequencing. Due to the position of the ApaIrestriction site, any amplification of a variable region for thispurpose has to include the first 11 nucleotides of the sequence of thehuman gamma-1 constant region in addition to the sequence of the ApaIsite. The sequence of the thus amplified human gamma-1 heavy chainconstant region inserted into the vector is listed as SEQ ID NO:45, theamino acid sequence of the thus expressed human gamma-1 constant regionis listed as SEQ ID NO: 46.

TABLE 4 mouse hybridoma cell lines used for antibody cloning clone mAbIsotype variable region oligomer pair in PCR chimerized antibody heavy43A 11 182-D1106-062 IgG2a SEQ ID NO: 55, 132 SEQ ID NO: 70, 71 SEQ IDNO: 100, 115 chain 163E12 182-D1106-294 IgG3 SEQ ID NO: 56, 133 SEQ IDNO: 72, 73 SEQ ID NO: 101, 116 125E1 182-D1106-279 IgG2a SEQ ID NO: 57,134 SEQ ID NO: 74, 75 SEQ ID NO: 102, 117 166E2 182-D1106-308 IgG3 SEQID NO: 59, 136 SEQ ID NO: 78, 79 SEQ ID NO: 104, 119 175D10182-D1106-362 IgG1 SEQ ID NO: ,58, 135 SEQ ID NO: 76, 77 SEQ ID NO: 103,118 45C1 182-D758-187 IgG2a SEQ ID NO: 60, 137 SEQ ID NO: 80, 81 SEQ IDNO: 105, 120 light 43A11 182-D1106-062 IgK SEQ ID NO: 62, 139 SEQ ID NO:84, 85 SEQ ID NO: 107, 122 chain 163E12 182-D1106-294 IgK SEQ ID NO: 61,138 SEQ ID NO: 82, 83 SEQ ID NO: 106, 121 125E1 182-D1106-279 IgK SEQ IDNO: 63, 140 SEQ ID NO: 86, 87 SEQ ID NO: 108, 123 166E2 182-D1106-308IgK SEQ ID NO: 66, 143 SEQ ID NO: 92, 93 SEQ ID NO: 111, 126 175D10182-D1106-362 IgK SEQ ID NO: 65, 142 SEQ ID NO: 90, 91 SEQ ID NO: 110,125 45C1 182-D758-187 IgK SEQ ID NO: 64, 141 SEQ ID NO: 88, 89 SEQ IDNO: 109, 124 45C1 182-D758-187 IgK SEQ ID NO: 67, 144 SEQ ID NO: 94, 95SEQ ID NO: 112, 127 45C1 182-D758-187 IgK SEQ ID NO: 68, 145 SEQ ID NO:96, 97 SEQ ID NO: 113, 128 45C1 182-D758-187 IgK SEQ ID NO: 69, 146 SEQID NO: 98, 99 SEQ ID NO: 114, 129

Corresponding to their murine counterparts the chimeric monoclonalantibodies were named adding the prefix “ch-” e.g. ch-43A11, ch-163E12,ch-125E1, ch-166E2, ch-175D10, ch-45C1.

Amplification of the murine variable regions of light and heavy chainswas carried out according to the “step-out PCR” method described in Matzet al. (Nucleic Acids Research, 1999, Vol. 27, No. 6). For this, totalRNA was prepared from monoclonal hybridoma cell lines (see Tab. 4) bystandard methods known to those skilled in the art, for example with theuse of RNeasy Mini Kit (Qiagen). Single stranded cDNA was preparedaccording to the “template-switch” method also described in Matz et al.(Nucleic Acids Research, 1999, Vol. 27, No. 6, 1558). In addition to an(dT)30 oligomer (SEQ ID NO: 47), it included a DNA/RNA hybrid oligomer(SEQ ID NO: 48) serving as an 5′ adaptor for template switching duringpolymerization of the cDNA strand. In this adaptor oligomer the lastthree nucleotides were ribo-instead of deoxyribonucleotides. Thesubsequent “step-out PCR” used an antisense oligomer targeted to theconstant region of the mouse kappa chain or to the constant region ofthe subclasses 1, 2a or 3 of the gamma chains (SEQ ID NO: 49 to 52,respectively). The IgG subclass of the murine monoclonal antibodyproduced by the hybridoma cell lines was afore immunologically analyzedwith IsoStrip (see Example 1), and the appropriate antisense oligomerwas chosen accordingly (see Tab. 4). A primer mix served as the senseoligomer in the “step-out PCR”, comprising the two oligomers listed inSEQ ID NO: 53 and 54. Some hybridoma cell lines expressed more than oneheavy or light chain (in addition to the chains expressed by the myelomacell line used for the generation of hybridomas). Table 4 summarizes theSEQ ID NOs of the cloned and sequenced variable regions of the murineantibody chains (SEQ ID NO: 55 to 69 and SEQ ID NO: 132 to 146) and ofthe cloned and sequenced full-length chimeric antibody chains (SEQ IDNO: 100 to 129).

The identified murine variable regions were then amplified by PCRomitting the 5′ UTR. and the 3′ mouse constant region, addingrestriction sites to the ends which allowed subcloning into the preparedexpression vectors carrying the human constant regions. In addition, thesense oligomers provided a consensus Kozak sequence (5′-GCCGCCACC-3′ or5′-AGCCACC-3′) and the antisense oligomers for heavy chain variableregions included the first 11 nucleotides of the human gamma-1 constantregion in addition to the ApaI restriction site (see Tab. 4, SEQ ID NO:70 to 99). Kappa light chain variable regions were cloned using HindIIIand BsiWI restriction enzymes, gamma heavy chain variable regionsdemanded HindIII and ApaI restriction enzymes. The heavy gamma chainvariable region of monoclonal antibody 45C1 contained an internalHindIII restriction site—here, the compatible BsaI enzyme was usedinstead (see SEQ ID NO: 80). SEQ ID NO: 100 to 114 show the nucleic acidsequences of the resulting chimerized antibodies (see Tab. 4). SEQ IDNO: 115 to 129 show the amino acid sequences of the accordinglyexpressed chimerized antibodies (see Tab. 4).

b. Generation and Production of Chimeric Antibodies Against CLD18

Mammalian cell lines producing chimeric antibodies with CLD18specificity were generated. The cell lines derived from HEK293T cells(ATCC CRL-11268). One day before transfection, 2.5×10⁷ cells were platedin a 14.5 cm tissue culture dish and cultured in 20 ml of completemedium, or alternatively 1×10⁷ cells were plated in a 10 cm tissueculture dish and cultured in 10 ml of complete medium, or alternatively0.6×10⁶ cells were plated in a well of a 12-well tissue plate andcultured in 2-3 ml of complete medium (complete medium: DMEM:F12 mediumsupplemented with 10% FBS without antibiotics). The recommended celldensity at the time of transfection should be 90% confluence.Immediately before transfection, medium wag replaced by fresh medium.HEK293T cells were transfected with transfection reagents, e.g.Lipofectamine 2000 (Invitrogen, 11668-019) or alternativelyPolyethylenimine (Sigma-Aldrich, 408727). Exemplified for transfectionof HEK293T cells a total DNA amount of 110 μg or 296 μg was used for a14.5 cm tissue dish, and the ratio of transfection agent to DNA was1:2.5 and 1:12 for Lipofectamine 2000 and PEI, respectively. 24 h aftertransfection medium was replaced with a GMP suitable medium, e.g. X-Vivo15 (Cambrex) or a chemical defined medium like Pro293a (Cambrex) withoutserum. Transfected HEK293T cells producing chimeric monoclonalantibodies against CLD18 were cultured for further 96 h. Crudesupernatants were harvested, sterile filtered and purified by proteinA-sepharose. Antibody concentration was determined by BCA Assay andpurity checked by sodium dodecylsulphate gel electrophoresis andcoomassie staining.

c. Binding Characteristics of Chimeric Monoclonal Antibodies

Binding specificity of the cloned and generated chimeric monoclonalantibodies to CLD18A2 was analyzed by flow cytometry as described inExample 3. HEK293 living cells stably expressing human CLD18A2(HEK293-CLD18A2) and HEK293 cells stably expressing human CLD18A1 (SEQID NOs: 7, 8) (HEK293-CLD18A1) were incubated for 30 min. at 4° C. withpurified HEK293T cell culture supernatants containing chimericmonoclonal antibodies, followed by incubation with APC-conjugatedF(ab′)₂ fragment goat anti-human IgG Fcγ secondary antibody andcounterstained with PI. Binding was assessed by flow cytometry using aBD FACSArray.

Similarly, endogenously CLD18A2 expressing human tumor cell lines, forexample KATO-III and NUGC-4 cells, were analyzed by flow cytometry.

FIGS. 31A and B show flow cytometric analyses of chimeric antibodiesch-43A11, ch-125E1, ch-163E12, ch-166E2, and ch-175D10. All of them shownative epitope recognition and exhibit specific and strong binding toCLD18A2 but not CLD18A1 expressing cells.

d. Complement Dependent Cytotoxicity (CDC)

Serum for complement lysis was prepared by drawing blood from healthyvolunteers into Serum-Monovette vacutainer tubes (Sarstedt, Nürmbrecht,Germany) which were then centrifuged at 600 g for 20 min. Serum washarvested and stored at −20° C. Control serum was heat inactivated at56° C. for 30 min before storage.

Protein A-sepharose-purified chimeric antibodies of this invention wereanalyzed for their capability to induce complement dependentcytotoxicity (CDC) against KATO-III cells endogenously expressing humanCLD18A2, as well as stably transfected CHO-CLD18A2 cells. Cells wereincubated with monoclonal antibodies ch-163E12, ch-166E2, and ch-175D10,respectively, in a final concentration of 2.5 μg/ml to 35 μg/ml for 30min. at 37° C. 20% human serum in RPMI was added to the cells andincubated for another 30 min. at 37° C. Thereafter, dead and livingcells were discriminated by PI staining in a final concentration of 2.5μg/ml and percentage of antibody-mediated cell lysis was determined byflow cytometry. For flow cytometric analysis a BD FACSArray flowcytometer was used (BD Biosciences, Mountain View, Calif.). At least10000 events were collected for analysis with cell debris excluded byadjustment of the forward sideward scatter (FSC/SSC) threshold. Specificlysis was calculated by the following formula: specific lysis=(%PT-positive cells in sample−% PI-positive cells in sample with heatinactivated serum). Specific lysis mediated by CDC was shown for severalantibodies. All three antibodies mediated robust CDC on CHO-CLD18A2cells (FIG. 32). On KATO-III cells antibodies ch-163E12 and ch-175D10were inducers of robust CDC.

e. Antibody-Dependent Cellular Cytotoxicity (ADCC)

FPLC-purified, chimeric antibodies of the invention were analyzed fortheir capability to induce antibody-dependent cellular cytotoxicity(ADCC) against KATO-III cells endogenously expressing human CLD18A2.

Human blood from healthy donors was diluted twice in phosphate buffer(PBS) and blood cells were layered on Ficoll (1077 g/ml, Pharmacia).After centrifugation, peripheral blood mononuclear cells (PBMC) werecollected from the interphase, washed and resuspended in X-Vivo-15culture medium supplemented with 5% heat-inactivated human serum.

15 h before the assay, KATO-III cells were transfected with luciferaseand plated at 5×10⁴ cells/well in a white microplate.

For the assay, effector cells (PBMC, prepared as described above) at aneffector to target (E:T) ratio of 20:1 and FPLC-purified chimericantibodies were added and incubated for 2-3 h at 37° C., 5%. CO₂. Finalconcentration of the antibody in the well was 50 μg/ml. After 2-3 h ofpre-incubation, lucifer yellow (BD Biosciences, San Jose USA) was addedat 1 mg/ml. Luminescence resulting from the oxidation of lucifer yellowby the luciferase of viable cells was measured continually for up to 6 husing a microplate-reader (Infinite200, Tecan, Switzerland). Percentageof cellular cytotoxicity was calculated using the following formula: %specific lysis=100−((sample luminescence counts−spontaneous luminescencecounts)/(maximal luminescence counts−spontaneous luminescencecounts)×100), with the spontaneous luminescence determined by addingTriton X-100 (0.2% final concentration), and the maximal signal measuredin the absence of antibodies.

Using this assay it was shown that monoclonal antibodies ch-163E12 andch-175D10 mediate strong ADCC on KATO-III cells (FIG. 33).

f. Proliferation Inhibition

FPLC-purified chimeric antibodies of the invention were analyzed fortheir capability to inhibit cell growth of KATO-III cells endogenouslyexpressing human CLD 18A2.

Target cells (KATO-111) were cultured in the presence of chimericrespective antibodies (see proliferation inhibition of murineantibodies, Example 7). FPLC purified chimeric antibodies ch-163E12 andch-166E2 were shown to inhibit cell proliferation.

10. Selection of Antibodies as Clinical Lead Candidates

Ideal clinical leads may cover a wide range of therapeutic anddiagnostic applications (see also section IV—Uses and Methods of theInvention). According to the invention antibodies directed to CLD18-A2are provided. It is shown that the antibodies provided according to theinvention offer a broad spectrum of properties regarding specificity,ability to induce CDC and ADCC and inhibit proliferation of cellsexpressing CLD18, in particular tumor cells. Furthermore, it has beendemonstrated that chimerisation of antibodies may lead to theacquisition of additional Fc-dependent effector functions not present inthe parental murine molecule. For example, it is shown herein thatantibody 175D10 with murine IgG1 does not induce complement dependentcytotoxicity (see Example 5), while ch-175D10 with human IgG1 inducesspecific lysis of constitutively CLD18 expressing tumor cells (see Tab.5 and Tab. 6).

Antibodies provided according to the present invention may becategorized into distinct classes according to their binding propertiesand their ability to mediate effector functions on cells expressingCLD18. From the antibodies provided according to the present invention,clinical lead candidates may be selected based on their functionalcharacteristics. An overview of properties for selected murine andchimeric antibodies of the invention is given in Tab. 5 and Tab. 6,respectively.

Clinical lead candidates of the invention may have one or more of thefollowing properties:

a) binding to human CLD18A2 but not to human CLD18A1 (e.g. 43A11, 45C1,125E1, 163E12, 166E2 and 175D10, and ch-43A11, ch-45C1, ch-125E1,ch-163E12, ch-166E2 and ch-175D10). For examples, see FIGS. 6A and 6B.

b) binding to mouse CLD18A2 but not to mouse CLD18A1 (e.g. 125E1,163E12, 166E2 and 175D10). For examples, see FIGS. 15A and 15B.

c) binding to CLD18 naturally expressed by tumor cells (e.g. 45C1,43A11, 125E1, 163E12, 166E2 and 175D10, and ch-45C1, ch-43A11, ch-125E1,ch-163E12, ch-166E2 and ch-175D10). For examples, see FIG. 13

d) binding to CLD18 in intercellular contact zones (e.g. 45C1, 43A11,125E1, 163E12, 166E2 and 1751310). For examples, see FIGS. 12A and 12B.

e) mediating CDC induced killing of cells, which express CLD18 (e.g.45C1, 125E1, 163E12, 166E2 and 175D10, and ch-163E12 and ch-175D10). Forexamples, see FIG. 32.

f) mediate ADCC induced killing of cells expressing CLD18 (e.g.ch-163E12 and ch-175D10). For examples, see FIG. 33.

g) inhibiting proliferation of cells expressing CLD18 (e.g. 45C1, 125E1,163E12, 166E2 and 175D10, and ch-163E12 and ch-166E2).

h) inhibiting tumor growth in xenograft models with cells expressingCLD18 (e.g. 43A11, 125E1, 163E12, 166E2, and 175D10). For examples, seeFIG. 24.

i) prolonging survival in xenograft models with cells expressing CLD18(e.g. 43A11, 125E1, 163E12, 166E2 and 175D10). For examples, see FIG.25B.

Exemplary Overview of Properties for Lead Candidate Selection

TABLE 5 murine antibodies binding binding binding of mediatinginhibiting inhibiting prolonging of human of mouse CLD18 on binding toCDC on proliferation tumor growth survival in CLD18A2 CLDl8A2 naturallyCLD18 in CLD18 of cells in xenograft xenograft but not but notexpressing contact expressing expressing expressing expressing antibodyA1 A1 tumor cells zones cells CLD18 CLD18 CLD18 45C1 + − + + (+) + (+)(+) 125E1 + + + + (+) + + + 163E12 + + + + + + + + 175D10 + + + + (+)(+) + + legend: + excellent perfounance, (+) performance in differentsetups.

TABLE 6 chimeric antibodies binding of binding of mediating mediatinginhibiting human CLD18 on CDC on ADCC on proliferation CLD18A naturallyCLD18 CLD18 of cells 2 but not expressing expressing expressingexpressing antibody Al turner cells cells cells CLD18 ch-45C1 + + n.d.n.d. n.d. ch-125E1 + + n.d. n.d. n.d. ch-163E12 + + + + +ch-175D10 + + + + n.d. legend: + excellent performance, (+) performancein different setups, n.d. not done.

The invention claimed is:
 1. An antibody having the ability of bindingto CLD18 and mediating killing of cells expressing CLD18, wherein theantibody binds to CLD18A2 but not to CLD18A1 and wherein the antibodybinds to an epitope within loopD3 consisting of SEQ ID NO: 29 ofCLD18A2, and said killing of cells is mediated by inducing CDC mediatedlysis and/or ADCC mediated lysis.
 2. The antibody of claim 1 whereinsaid killing of cells is induced by binding of said antibody to CLD18A2expressed by said cells.
 3. The antibody of claim 1 wherein said cellsexpressing CLD18A2 are cancer cells.
 4. The antibody of claim 3 whereinthe cancer cells are selected from the group consisting of tumorigenicgastric, esophageal, pancreatic and lung cancer cells.
 5. The antibodyof claim 1 wherein said ADCC mediated lysis takes place in the presenceof effector cells selected from the group consisting of monocytes,mononuclear cells, NK cells and PMNs.
 6. The antibody of claim 1 whichis a monoclonal, chimeric or humanized antibody, or an antigen-bindingfragment of an antibody.
 7. The antibody of claim 1 selected from thegroup consisting of an IgG1, an IgG2, preferably IgG2a and IgG2b, anIgG3, an IgG4, and an IgM antibody.
 8. The antibody of claim 1 whereinCLD18A2 has the amino acid sequence according to SEQ ID NO:2.
 9. Theantibody of claim 1 wherein CLD18A1 has the amino acid sequenceaccording to SEQ ID NO:8.
 10. The antibody of claim 1 which is specificfor cancer cells, preferably stomach cancer cells.
 11. The antibody ofclaim 1 wherein said CLD18A2 is expressed on the surface of said cells.12. The antibody of claim 1 which is obtainable by a method comprisingthe step of immunizing an animal with a protein or peptide having anamino acid sequence selected from the group consisting of SEQ ID NO: 2,4, 6, 18, 20, and 31, or an immunogenic fragment thereof, or a nucleicacid or host cell expressing said protein or peptide, or immunogenicfragment thereof.
 13. A hybridoma capable of producing the antibody ofclaim
 1. 14. A conjugate comprising an antibody of claim 1 coupled to atherapeutic agent.
 15. The conjugate of claim 14 wherein the therapeuticagent is a toxin, a radioisotope, a drug or a cytotoxic agent.
 16. Apharmaceutical composition comprising an antibody of claim 1, and apharmaceutically acceptable carrier.
 17. An antibody having the abilityof binding to CLD18 and mediating killing of cells expressing CLD18,wherein the antibody binds to CLD18A2 but not to CLD18A1 and wherein theantibody binds to the amino acid sequence of SEQ ID NO: 29, and saidkilling of cells is mediated by inducing CDC mediated lysis and/or ADCCmediated lysis.
 18. An antibody having the ability of binding to CLD18and mediating killing of cells expressing CLD18, wherein the antibodybinds to CLD18A2 but not to CLD18A1 and wherein the antibody binds to apeptide consisting of the amino acid sequence of SEQ ID NO: 29, and saidkilling of cells is mediated by inducing CDC mediated lysis and/or ADCCmediated lysis.
 19. A method of killing a cancer cell expressingCLD18A2, comprising contacting the cell with an effective amount of anantibody of claim
 1. 20. A method of treating cancer involving cellsexpressing CLD18A2, comprising administering to a subject an antibody ofclaim
 1. 21. The method of claim 20 wherein the cancer is selected fromthe group consisting of gastric cancer, esophageal cancer, pancreaticcancer, lung cancer, ovarian cancer, colon cancer, hepatic cancer,head-neck cancer, and cancer of the gallbladder.